| Abdelhakim AH, Salgado EN, Fu X, Pasham M, Nicastro D, Kirchhausen T and Harrison SC (2014), "Structural correlates of rotavirus cell entry.", PLoS Pathog. Vol. 10(9), pp. e1004355. |
| Abstract: Cell entry by non-enveloped viruses requires translocation into the cytosol of a macromolecular complex--for double-strand RNA viruses, a complete subviral particle. We have used live-cell fluorescence imaging to follow rotavirus entry and penetration into the cytosol of its ∼ 700 Å inner capsid particle ("double-layered particle", DLP). We label with distinct fluorescent tags the DLP and each of the two outer-layer proteins and track the fates of each species as the particles bind and enter BSC-1 cells. Virions attach to their glycolipid receptors in the host cell membrane and rapidly become inaccessible to externally added agents; most particles that release their DLP into the cytosol have done so by ∼ 10 minutes, as detected by rapid diffusional motion of the DLP away from residual outer-layer proteins. Electron microscopy shows images of particles at various stages of engulfment into tightly fitting membrane invaginations, consistent with the interpretation that rotavirus particles drive their own uptake. Electron cryotomography of membrane-bound virions also shows closely wrapped membrane. Combined with high resolution structural information about the viral components, these observations suggest a molecular model for membrane disruption and DLP penetration. |
BibTeX:
@article{Abdelhakim2014,
author = {Abdelhakim, Aliaa H. and Salgado, Eric N. and Fu, Xiaofeng and Pasham, Mithun and Nicastro, Daniela and Kirchhausen, Tomas and Harrison, Stephen C.},
title = {Structural correlates of rotavirus cell entry.},
journal = {PLoS Pathog},
school = {Laboratory of Molecular Medicine, Boston Children's Hospital and Harvard Medical School, Boston, Massachusetts, United States of America; Howard Hughes Medical Institute, Boston, Massachusetts, United States of America.},
year = {2014},
volume = {10},
number = {9},
pages = {e1004355},
url = {http://dx.doi.org/10.1371/journal.ppat.1004355},
doi = {10.1371/journal.ppat.1004355}
}
|
| Abrusci P, Vergara-Irigaray M, Johnson S, Beeby MD, Hendrixson DR, Roversi P, Friede ME, Deane JE, Jensen GJ, Tang CM and Lea SM (2013), "Architecture of the major component of the type III secretion system export apparatus.", Nat Struct Mol Biol. Vol. 20(1), pp. 99-104. |
| Abstract: Type III secretion systems (T3SSs) are bacterial membrane-embedded nanomachines designed to export specifically targeted proteins from the bacterial cytoplasm. Secretion through T3SS is governed by a subset of inner membrane proteins termed the 'export apparatus'. We show that a key member of the Shigella flexneri export apparatus, MxiA, assembles into a ring essential for secretion in vivo. The ring-forming interfaces are well-conserved in both nonflagellar and flagellar homologs, implying that the ring is an evolutionarily conserved feature in these systems. Electron cryo-tomography revealed a T3SS-associated cytoplasmic torus of size and shape corresponding to those of the MxiA ring aligned to the secretion channel located between the secretion pore and the ATPase complex. This defines the molecular architecture of the dominant component of the export apparatus and allows us to propose a model for the molecular mechanisms controlling secretion. |
BibTeX:
@article{Abrusci2013,
author = {Abrusci, Patrizia and Vergara-Irigaray, Marta and Johnson, Steven and Beeby, Morgan D. and Hendrixson, David R. and Roversi, Pietro and Friede, Miriam E. and Deane, Janet E. and Jensen, Grant J. and Tang, Christoph M. and Lea, Susan M.},
title = {Architecture of the major component of the type III secretion system export apparatus.},
journal = {Nat Struct Mol Biol},
school = {Sir William Dunn School of Pathology, Oxford University, Oxford, UK.},
year = {2013},
volume = {20},
number = {1},
pages = {99--104},
url = {http://dx.doi.org/10.1038/nsmb.2452},
doi = {10.1038/nsmb.2452}
}
|
| Althoff T, Davies KM, Schulze S, Joos F and Kühlbrandt W (2012), "GRecon: a method for the lipid reconstitution of membrane proteins.", Angew Chem Int Ed Engl. Vol. 51(33), pp. 8343-8347. |
BibTeX:
@article{Althoff2012,
author = {Althoff, Thorsten and Davies, Karen M. and Schulze, Sabrina and Joos, Friederike and Kühlbrandt, Werner},
title = {GRecon: a method for the lipid reconstitution of membrane proteins.},
journal = {Angew Chem Int Ed Engl},
school = {Max-Planck-Institut für Biophysik, Strukturbiologie, Max-von-Laue-Strasse 3, 60438 Frankfurt, Germany.},
year = {2012},
volume = {51},
number = {33},
pages = {8343--8347},
url = {http://dx.doi.org/10.1002/anie.201202094},
doi = {10.1002/anie.201202094}
}
|
| Auer GK, Oliver PM, Rajendram M, Lin T-Y, Yao Q, Jensen GJ and Weibel DB (2019), "Bacterial swarming reduces Proteus mirabilis and Vibrio parahaemolyticus cell stiffness and increases β-lactam susceptibility", bioRxiv., pp. 275941. Cold Spring Harbor Laboratory.
[BibTeX] |
BibTeX:
@article{Auer2019,
author = {Auer, George K and Oliver, Piercen M and Rajendram, Manohary and Lin, Ti-Yu and Yao, Qing and Jensen, Grant J and Weibel, Douglas B},
title = {Bacterial swarming reduces Proteus mirabilis and Vibrio parahaemolyticus cell stiffness and increases β-lactam susceptibility},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
year = {2019},
pages = {275941}
}
|
| Awata J, Song K, Lin J, King SM, Sanderson MJ, Nicastro D and Witman GB (2015), "DRC3 connects the N-DRC to dynein g to regulate flagellar waveform.", Mol Biol Cell. Vol. 26(15), pp. 2788-2800. |
| Abstract: The nexin-dynein regulatory complex (N-DRC), which is a major hub for the control of flagellar motility, contains at least 11 different subunits. A major challenge is to determine the location and function of each of these subunits within the N-DRC. We characterized a Chlamydomonas mutant defective in the N-DRC subunit DRC3. Of the known N-DRC subunits, the drc3 mutant is missing only DRC3. Like other N-DRC mutants, the drc3 mutant has a defect in flagellar motility. However, in contrast to other mutations affecting the N-DRC, drc3 does not suppress flagellar paralysis caused by loss of radial spokes. Cryo-electron tomography revealed that the drc3 mutant lacks a portion of the N-DRC linker domain, including the L1 protrusion, part of the distal lobe, and the connection between these two structures, thus localizing DRC3 to this part of the N-DRC. This and additional considerations enable us to assign DRC3 to the L1 protrusion. Because the L1 protrusion is the only non-dynein structure in contact with the dynein g motor domain in wild-type axonemes and this is the only N-DRC-dynein connection missing in the drc3 mutant, we conclude that DRC3 interacts with dynein g to regulate flagellar waveform. |
BibTeX:
@article{Awata2015,
author = {Awata, Junya and Song, Kangkang and Lin, Jianfeng and King, Stephen M. and Sanderson, Michael J. and Nicastro, Daniela and Witman, George B.},
title = {DRC3 connects the N-DRC to dynein g to regulate flagellar waveform.},
journal = {Mol Biol Cell},
school = {Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, MA 01655 nicastro@brandeis.edu george.witman@umassmed.edu.},
year = {2015},
volume = {26},
number = {15},
pages = {2788--2800},
url = {http://dx.doi.org/10.1091/mbc.E15-01-0018},
doi = {10.1091/mbc.E15-01-0018}
}
|
| Azubel M, Carter SD, Weiszmann J, Zhang J, Jensen GJ, Li Y and Kornberg RD (2019), "FGF21 trafficking in intact human cells revealed by cryo-electron tomography with gold nanoparticles", eLife. Vol. 8, pp. e43146. eLife Sciences Publications Limited.
[BibTeX] |
BibTeX:
@article{Azubel2019,
author = {Azubel, Maia and Carter, Stephen D and Weiszmann, Jennifer and Zhang, Jun and Jensen, Grant J and Li, Yang and Kornberg, Roger D},
title = {FGF21 trafficking in intact human cells revealed by cryo-electron tomography with gold nanoparticles},
journal = {eLife},
publisher = {eLife Sciences Publications Limited},
year = {2019},
volume = {8},
pages = {e43146}
}
|
| Azubel M, Koh AL, Koyasu K, Tsukuda T and Kornberg RD (2017), "Structure determination of a water-soluble 144-gold atom particle at atomic resolution by aberration-corrected electron microscopy", ACS nano. Vol. 11(12), pp. 11866-11871. ACS Publications.
[BibTeX] |
BibTeX:
@article{Azubel2017,
author = {Azubel, Maia and Koh, Ai Leen and Koyasu, Kiichirou and Tsukuda, Tatsuya and Kornberg, Roger D},
title = {Structure determination of a water-soluble 144-gold atom particle at atomic resolution by aberration-corrected electron microscopy},
journal = {ACS nano},
publisher = {ACS Publications},
year = {2017},
volume = {11},
number = {12},
pages = {11866--11871}
}
|
| Azubel M, Koivisto J, Malola S, Bushnell D, Hura GL, Koh AL, Tsunoyama H, Tsukuda T, Pettersson M, Häkkinen H and others (2014), "Electron microscopy of gold nanoparticles at atomic resolution", Science. Vol. 345(6199), pp. 909-912. American Association for the Advancement of Science.
[BibTeX] |
BibTeX:
@article{Azubel2014,
author = {Azubel, Maia and Koivisto, Jaakko and Malola, Sami and Bushnell, David and Hura, Greg L and Koh, Ai Leen and Tsunoyama, Hironori and Tsukuda, Tatsuya and Pettersson, Mika and Häkkinen, Hannu and others},
title = {Electron microscopy of gold nanoparticles at atomic resolution},
journal = {Science},
publisher = {American Association for the Advancement of Science},
year = {2014},
volume = {345},
number = {6199},
pages = {909--912}
}
|
| Azubel M and Kornberg RD (2016), "Synthesis of water-soluble, thiolate-protected gold nanoparticles uniform in size", Nano letters. Vol. 16(5), pp. 3348-3351. ACS Publications.
[BibTeX] |
BibTeX:
@article{Azubel2016,
author = {Azubel, Maia and Kornberg, Roger D},
title = {Synthesis of water-soluble, thiolate-protected gold nanoparticles uniform in size},
journal = {Nano letters},
publisher = {ACS Publications},
year = {2016},
volume = {16},
number = {5},
pages = {3348--3351}
}
|
| Azubel M and Kornberg RD (2017), "Synthesis of water-soluble thiolate-protected gold nanoparticles of uniform size and conjugates thereof". October 26, 2017.
[BibTeX] |
BibTeX:
@misc{Azubel2017a,
author = {Azubel, Maia and Kornberg, Roger D},
title = {Synthesis of water-soluble thiolate-protected gold nanoparticles of uniform size and conjugates thereof},
publisher = {Google Patents},
year = {2017},
note = {US Patent App. 15/493,726}
}
|
| Azubel M, Wolf SG, Sperling J and Sperling R (2004), "Three-dimensional structure of the native spliceosome by cryo-electron microscopy", Molecular cell. Vol. 15(5), pp. 833-839. Elsevier.
[BibTeX] |
BibTeX:
@article{Azubel2004,
author = {Azubel, Maia and Wolf, Sharon G and Sperling, Joseph and Sperling, Ruth},
title = {Three-dimensional structure of the native spliceosome by cryo-electron microscopy},
journal = {Molecular cell},
publisher = {Elsevier},
year = {2004},
volume = {15},
number = {5},
pages = {833--839}
}
|
| Bai X-c, Rajendra E, Yang G, Shi Y and Scheres SHW (2015), "Sampling the conformational space of the catalytic subunit of human γ-secretase.", eLife. Vol. 4 |
| Abstract: Human γ-secretase is an intra-membrane protease that cleaves many different substrates. Aberrant cleavage of Notch is implicated in cancer, while abnormalities in cutting amyloid precursor protein lead to Alzheimer's disease. Our previous cryo-EM structure of γ-secretase revealed considerable disorder in its catalytic subunit presenilin. Here, we describe an image classification procedure that characterizes molecular plasticity at the secondary structure level, and apply this method to identify three distinct conformations in our previous sample. In one of these conformations, an additional transmembrane helix is visible that cannot be attributed to the known components of γ-secretase. In addition, we present a γ-secretase structure in complex with the dipeptidic inhibitor N-[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT). Our results reveal how conformational mobility in the second and sixth transmembrane helices of presenilin is greatly reduced upon binding of DAPT or the additional helix, and form the basis for a new model of how substrate enters the transmembrane domain. |
BibTeX:
@article{Bai2015,
author = {Bai, Xiao-chen and Rajendra, Eeson and Yang, Guanghui and Shi, Yigong and Scheres, Sjors H W},
title = {Sampling the conformational space of the catalytic subunit of human γ-secretase.},
journal = {eLife},
year = {2015},
volume = {4},
doi = {10.7554/eLife.11182}
}
|
| Baker LA, Sinnige T, Schellenberger P, de Keyzer J, Siebert CA, Driessen AJM, Baldus M and Grünewald K (2018), "Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments.", Structure (London, England : 1993). Vol. 26, pp. 161-170.e3. |
| Abstract: Membrane proteins remain challenging targets for structural biology, despite much effort, as their native environment is heterogeneous and complex. Most methods rely on detergents to extract membrane proteins from their native environment, but this removal can significantly alter the structure and function of these proteins. Here, we overcome these challenges with a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample. Our method allows the structure and function of membrane proteins to be studied in their native environments, across different spatial and temporal resolutions, and the combination is more powerful than each technique individually. We use the method to demonstrate that the bacterial membrane protein YidC adopts a different conformation in native membranes and that substrate binding to YidC in these native membranes differs from purified and reconstituted systems. |
BibTeX:
@article{Baker2018,
author = {Baker, Lindsay A and Sinnige, Tessa and Schellenberger, Pascale and de Keyzer, Jeanine and Siebert, C Alistair and Driessen, Arnold J M and Baldus, Marc and Grünewald, Kay},
title = {Combined 1H-Detected Solid-State NMR Spectroscopy and Electron Cryotomography to Study Membrane Proteins across Resolutions in Native Environments.},
journal = {Structure (London, England : 1993)},
year = {2018},
volume = {26},
pages = {161--170.e3},
doi = {10.1016/j.str.2017.11.011}
}
|
| Baldwin PR, Tan YZ, Eng ET, Rice WJ, Noble AJ, Negro CJ, Cianfrocco MA, Potter CS and Carragher B (2018), "Big data in cryoEM: automated collection, processing and accessibility of EM data.", Current opinion in microbiology. Vol. 43, pp. 1-8. |
| Abstract: The scope and complexity of cryogenic electron microscopy (cryoEM) data has greatly increased, and will continue to do so, due to recent and ongoing technical breakthroughs that have led to much improved resolutions for macromolecular structures solved using this method. This big data explosion includes single particle data as well as tomographic tilt series, both generally acquired as direct detector movies of ∼10-100 frames per image or per tilt-series. We provide a brief survey of the developments leading to the current status, and describe existing cryoEM pipelines, with an emphasis on the scope of data acquisition, methods for automation, and use of cloud storage and computing. |
BibTeX:
@article{Baldwin2018,
author = {Baldwin, Philip R. and Tan, Yong Zi and Eng, Edward T. and Rice, William J. and Noble, Alex J. and Negro, Carl J. and Cianfrocco, Michael A. and Potter, Clinton S. and Carragher, Bridget},
title = {Big data in cryoEM: automated collection, processing and accessibility of EM data.},
journal = {Current opinion in microbiology},
year = {2018},
volume = {43},
pages = {1--8},
doi = {10.1016/j.mib.2017.10.005}
}
|
| Barber CF, Heuser T, Carbajal-González BI, Botchkarev Jr VV and Nicastro D (2012), "Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella.", Mol Biol Cell. Vol. 23(1), pp. 111-120. |
| Abstract: Radial spokes (RSs) play an essential role in the regulation of axonemal dynein activity and thus of ciliary and flagellar motility. However, few details are known about the complexes involved. Using cryo-electron tomography and subtomogram averaging, we visualized the three-dimensional structure of the radial spokes in Chlamydomonas flagella in unprecedented detail. Unlike many other species, Chlamydomonas has only two spokes per axonemal repeat, RS1 and RS2. Our data revealed previously uncharacterized features, including two-pronged spoke bases that facilitate docking to the doublet microtubules, and that inner dyneins connect directly to the spokes. Structures of wild type and the headless spoke mutant pf17 were compared to define the morphology and boundaries of the head, including a direct RS1-to-RS2 interaction. Although the overall structures of the spokes are very similar, we also observed some differences, corroborating recent findings about heterogeneity in the docking of RS1 and RS2. In place of a third radial spoke we found an uncharacterized, shorter electron density named "radial spoke 3 stand-in," which structurally bears no resemblance to RS1 and RS2 and is unaltered in the pf17 mutant. These findings demonstrate that radial spokes are heterogeneous in structure and may play functionally distinct roles in axoneme regulation. |
BibTeX:
@article{Barber2012,
author = {Barber, Cynthia F. and Heuser, Thomas and Carbajal-González, Blanca I. and Botchkarev, Jr, Vladimir V and Nicastro, Daniela},
title = {Three-dimensional structure of the radial spokes reveals heterogeneity and interactions with dyneins in Chlamydomonas flagella.},
journal = {Mol Biol Cell},
school = {Biology Department, Rosenstiel Center, Brandeis University, Waltham, MA 02454, USA.},
year = {2012},
volume = {23},
number = {1},
pages = {111--120},
url = {http://dx.doi.org/10.1091/mbc.E11-08-0692},
doi = {10.1091/mbc.E11-08-0692}
}
|
| Basanta B, Chowdhury S, Lander GC and Grotjahn DA (2020), "A guided approach for subtomogram averaging of challenging macromolecular assemblies.", Journal of structural biology: X. Vol. 4, pp. 100041. |
BibTeX:
@article{Basanta2020,
author = {Basanta, Benjamin and Chowdhury, Saikat and Lander, Gabriel C. and Grotjahn, Danielle A.},
title = {A guided approach for subtomogram averaging of challenging macromolecular assemblies.},
journal = {Journal of structural biology: X},
year = {2020},
volume = {4},
pages = {100041},
doi = {10.1016/j.yjsbx.2020.100041}
}
|
| Basler M, Pilhofer M, Henderson GP, Jensen GJ and Mekalanos JJ (2012), "Type VI secretion requires a dynamic contractile phage tail-like structure.", Nature. Vol. 483(7388), pp. 182-186. |
| Abstract: Type VI secretion systems are bacterial virulence-associated nanomachines composed of proteins that are evolutionarily related to components of bacteriophage tails. Here we show that protein secretion by the type VI secretion system of Vibrio cholerae requires the action of a dynamic intracellular tubular structure that is structurally and functionally homologous to contractile phage tail sheath. Time-lapse fluorescence light microscopy reveals that sheaths of the type VI secretion system cycle between assembly, quick contraction, disassembly and re-assembly. Whole-cell electron cryotomography further shows that the sheaths appear as long tubular structures in either extended or contracted conformations that are connected to the inner membrane by a distinct basal structure. These data support a model in which the contraction of the type VI secretion system sheath provides the energy needed to translocate proteins out of effector cells and into adjacent target cells. |
BibTeX:
@article{Basler2012,
author = {Basler, M. and Pilhofer, M. and Henderson, G. P. and Jensen, G. J. and Mekalanos, J. J.},
title = {Type VI secretion requires a dynamic contractile phage tail-like structure.},
journal = {Nature},
school = {Department of Microbiology and Immunobiology, Harvard Medical School, 200 Longwood Avenue, Boston, Massachusetts 02115, USA.},
year = {2012},
volume = {483},
number = {7388},
pages = {182--186},
url = {http://dx.doi.org/10.1038/nature10846},
doi = {10.1038/nature10846}
}
|
| Basta T, Wu H-J, Morphew MK, Lee J, Ghosh N, Lai J, Heumann JM, Wang K, Lee YC, Rees DC and Stowell MHB (2014), "Self-assembled lipid and membrane protein polyhedral nanoparticles.", Proc Natl Acad Sci U S A. Vol. 111(2), pp. 670-674. |
| Abstract: We demonstrate that membrane proteins and phospholipids can self-assemble into polyhedral arrangements suitable for structural analysis. Using the Escherichia coli mechanosensitive channel of small conductance (MscS) as a model protein, we prepared membrane protein polyhedral nanoparticles (MPPNs) with uniform radii of ∼ 20 nm. Electron cryotomographic analysis established that these MPPNs contain 24 MscS heptamers related by octahedral symmetry. Subsequent single-particle electron cryomicroscopy yielded a reconstruction at ∼ 1-nm resolution, revealing a conformation closely resembling the nonconducting state. The generality of this approach has been addressed by the successful preparation of MPPNs for two unrelated proteins, the mechanosensitive channel of large conductance and the connexon Cx26, using a recently devised microfluidics-based free interface diffusion system. MPPNs provide not only a starting point for the structural analysis of membrane proteins in a phospholipid environment, but their closed surfaces should facilitate studies in the presence of physiological transmembrane gradients, in addition to potential applications as drug delivery carriers or as templates for inorganic nanoparticle formation. |
BibTeX:
@article{Basta2014,
author = {Basta, Tamara and Wu, Hsin-Jui and Morphew, Mary K. and Lee, Jonas and Ghosh, Nilanjan and Lai, Jeffrey and Heumann, John M. and Wang, Keeshia and Lee, Y. C. and Rees, Douglas C. and Stowell, Michael H B.},
title = {Self-assembled lipid and membrane protein polyhedral nanoparticles.},
journal = {Proc Natl Acad Sci U S A},
school = {Molecular, Cellular, and Developmental Biology and Mechanical Engineering, University of Colorado at Boulder, Boulder, CO 80309.},
year = {2014},
volume = {111},
number = {2},
pages = {670--674},
url = {http://dx.doi.org/10.1073/pnas.1321936111},
doi = {10.1073/pnas.1321936111}
}
|
| Bazan R, Schröfel A, Joachimiak E, Poprzeczko M, Pigino G and Wloga D (2021), "Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link.", PLoS genetics. Vol. 17, pp. e1009388. |
| Abstract: Ciliary beating requires the coordinated activity of numerous axonemal complexes. The protein composition and role of radial spokes (RS), nexin links (N-DRC) and dyneins (ODAs and IDAs) is well established. However, how information is transmitted from the central apparatus to the RS and across other ciliary structures remains unclear. Here, we identify a complex comprising the evolutionarily conserved proteins Ccdc96 and Ccdc113, positioned parallel to N-DRC and forming a connection between RS3, dynein g, and N-DRC. Although Ccdc96 and Ccdc113 can be transported to cilia independently, their stable docking and function requires the presence of both proteins. Deletion of either CCDC113 or CCDC96 alters cilia beating frequency, amplitude and waveform. We propose that the Ccdc113/Ccdc96 complex transmits signals from RS3 and N-DRC to dynein g and thus regulates its activity and the ciliary beat pattern. |
BibTeX:
@article{Bazan2021,
author = {Bazan, Rafał and Schröfel, Adam and Joachimiak, Ewa and Poprzeczko, Martyna and Pigino, Gaia and Wloga, Dorota},
title = {Ccdc113/Ccdc96 complex, a novel regulator of ciliary beating that connects radial spoke 3 to dynein g and the nexin link.},
journal = {PLoS genetics},
year = {2021},
volume = {17},
pages = {e1009388},
doi = {10.1371/journal.pgen.1009388}
}
|
| Beeby M, Ribardo DA, Brennan CA, Ruby EG, Jensen GJ and Hendrixson DR (2016), "Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.", Proc Natl Acad Sci U S A. Vol. 113(13), pp. E1917-E1926. |
| Abstract: Although it is known that diverse bacterial flagellar motors produce different torques, the mechanism underlying torque variation is unknown. To understand this difference better, we combined genetic analyses with electron cryo-tomography subtomogram averaging to determine in situ structures of flagellar motors that produce different torques, fromCampylobacterandVibriospecies. For the first time, to our knowledge, our results unambiguously locate the torque-generating stator complexes and show that diverse high-torque motors use variants of an ancestrally related family of structures to scaffold incorporation of additional stator complexes at wider radii from the axial driveshaft than in the model enteric motor. We identify the protein components of these additional scaffold structures and elucidate their sequential assembly, demonstrating that they are required for stator-complex incorporation. These proteins are widespread, suggesting that different bacteria have tailored torques to specific environments by scaffolding alternative stator placement and number. Our results quantitatively account for different motor torques, complete the assignment of the locations of the major flagellar components, and provide crucial constraints for understanding mechanisms of torque generation and the evolution of multiprotein complexes. |
BibTeX:
@article{Beeby2016,
author = {Beeby, Morgan and Ribardo, Deborah A. and Brennan, Caitlin A. and Ruby, Edward G. and Jensen, Grant J. and Hendrixson, David R.},
title = {Diverse high-torque bacterial flagellar motors assemble wider stator rings using a conserved protein scaffold.},
journal = {Proc Natl Acad Sci U S A},
school = {Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390; mbeeby@imperial.ac.uk david.hendrixson@utsouthwestern.edu.},
year = {2016},
volume = {113},
number = {13},
pages = {E1917--E1926},
url = {http://dx.doi.org/10.1073/pnas.1518952113},
doi = {10.1073/pnas.1518952113}
}
|
| Bollschweiler D, Radu L, Joudeh L, Plitzko JM, Henderson RM, Mela I and Pellegrini L (2019), "Molecular architecture of the SYCP3 fibre and its interaction with DNA.", Open biology. Vol. 9, pp. 190094. |
| Abstract: The synaptonemal complex (SC) keeps homologous chromosomes in close alignment during meiotic recombination. A hallmark of the SC is the presence of its constituent protein SYCP3 on the chromosome axis. During SC assembly, SYCP3 is deposited on both axes of the homologue pair, forming axial elements that fuse into the lateral element (LE) in the tripartite structure of the mature SC. We have used cryo-electron tomography and atomic force microscopy to study the mechanism of assembly and DNA binding of the SYCP3 fibre. We find that the three-dimensional architecture of the fibre is built on a highly irregular arrangement of SYCP3 molecules displaying very limited local geometry. Interaction between SYCP3 molecules is driven by the intrinsically disordered tails of the protein, with no contact between the helical cores, resulting in a flexible fibre assembly. We demonstrate that the SYCP3 fibre can engage in extensive interactions with DNA, indicative of an efficient mechanism for incorporation of DNA within the fibre. Our findings suggest that SYCP3 deposition on the chromosome axis might take place by polymerization into a fibre that is fastened to the chromosome surface via DNA binding. |
BibTeX:
@article{Bollschweiler2019,
author = {Bollschweiler, Daniel and Radu, Laura and Joudeh, Luay and Plitzko, Jürgen M and Henderson, Robert M and Mela, Ioanna and Pellegrini, Luca},
title = {Molecular architecture of the SYCP3 fibre and its interaction with DNA.},
journal = {Open biology},
year = {2019},
volume = {9},
pages = {190094},
doi = {10.1098/rsob.190094}
}
|
| Bollschweiler D, Radu L and Pellegrini L (2018), "Cryo-electron tomography of SYCP3 fibers under native conditions.", Methods in cell biology. Vol. 145, pp. 347-371. |
| Abstract: The synaptonemal complex (SC) forms during the early stages of meiotic prophase I, when it mediates the pairing of homologous chromosomes. Despite the crucial role of the SC in chromosome synapsis and genetic recombination, the molecular details of its function are still unclear. High-resolution information on the structure of SC proteins would be very valuable to elucidate the molecular basis of their function in meiosis. Here we show how cryo-electron tomography and subtomographic averaging can be usefully applied to provide insights into the structure of the helical SYCP3 protein in its filamentous state. The establishment of such method should prove of use for structural studies of other SC proteins, such as SYCP1 and the TEX12-SYCE2 complex, which can form physiologically relevant filamentous assemblies, and ultimately for the structural analysis of the SC. |
BibTeX:
@article{Bollschweiler2018,
author = {Bollschweiler, Daniel and Radu, Laura and Pellegrini, Luca},
title = {Cryo-electron tomography of SYCP3 fibers under native conditions.},
journal = {Methods in cell biology},
year = {2018},
volume = {145},
pages = {347--371},
doi = {10.1016/bs.mcb.2018.03.034}
}
|
| Bower R, Tritschler D, Mills KV, Heuser T, Nicastro D and Porter ME (2018), "DRC2/CCDC65 is a central hub for assembly of the nexin--dynein regulatory complex and other regulators of ciliary and flagellar motility", Molecular biology of the cell. Vol. 29(2), pp. 137-153. Am Soc Cell Biol.
[BibTeX] |
BibTeX:
@article{Bower2018,
author = {Bower, Raqual and Tritschler, Douglas and Mills, Kristyn VanderWaal and Heuser, Thomas and Nicastro, Daniela and Porter, Mary E},
title = {DRC2/CCDC65 is a central hub for assembly of the nexin--dynein regulatory complex and other regulators of ciliary and flagellar motility},
journal = {Molecular biology of the cell},
publisher = {Am Soc Cell Biol},
year = {2018},
volume = {29},
number = {2},
pages = {137--153}
}
|
| Briegel A, Ames P, Gumbart JC, Oikonomou CM, Parkinson JS and Jensen GJ (2013), "The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state.", Mol Microbiol. Vol. 89(5), pp. 831-841. |
| Abstract: Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favourable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signalling arrays in well-defined kinase activity states. The arrays were well ordered in all signalling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, the phosphorylation site domain and the binding domain for response regulator target proteins. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signalling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signalling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains. |
BibTeX:
@article{Briegel2013,
author = {Briegel, Ariane and Ames, Peter and Gumbart, James C. and Oikonomou, Catherine M. and Parkinson, John S. and Jensen, Grant J.},
title = {The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state.},
journal = {Mol Microbiol},
school = {California Institute of Technology, 1200 E. California Blvd, Pasadena, CA, 91125, USA.},
year = {2013},
volume = {89},
number = {5},
pages = {831--841},
url = {http://dx.doi.org/10.1111/mmi.12309},
doi = {10.1111/mmi.12309}
}
|
| Briegel A, Beeby M, Thanbichler M and Jensen GJ (2011), "Activated chemoreceptor arrays remain intact and hexagonally packed.", Mol Microbiol. Vol. 82(3), pp. 748-757. |
| Abstract: Bacterial chemoreceptors cluster into exquisitively sensitive, tunable, highly ordered, polar arrays. While these arrays serve as paradigms of cell signalling in general, it remains unclear what conformational changes transduce signals from the periplasmic tips, where attractants and repellents bind, to the cytoplasmic signalling domains. Conflicting reports support and contest the hypothesis that activation causes large changes in the packing arrangement of the arrays, up to and including their complete disassembly. Using electron cryotomography, here we show that in Caulobacter crescentus, chemoreceptor arrays in cells grown in different media and immediately after exposure to the attractant galactose all exhibit the same 12 nm hexagonal packing arrangement, array size and other structural parameters. ΔcheB and ΔcheR mutants mimicking attractant- or repellent-bound states prior to adaptation also show the same lattice structure. We conclude that signal transduction and amplification must be accomplished through only small, nanoscale conformational changes. |
BibTeX:
@article{Briegel2011,
author = {Briegel, Ariane and Beeby, Morgan and Thanbichler, Martin and Jensen, Grant J.},
title = {Activated chemoreceptor arrays remain intact and hexagonally packed.},
journal = {Mol Microbiol},
school = {Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.},
year = {2011},
volume = {82},
number = {3},
pages = {748--757},
url = {http://dx.doi.org/10.1111/j.1365-2958.2011.07854.x},
doi = {10.1111/j.1365-2958.2011.07854.x}
}
|
| Briegel A, Li X, Bilwes AM, Hughes KT, Jensen GJ and Crane BR (2012), "Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins.", Proc Natl Acad Sci U S A. Vol. 109(10), pp. 3766-3771. |
| Abstract: Chemoreceptor arrays are supramolecular transmembrane machines of unknown structure that allow bacteria to sense their surroundings and respond by chemotaxis. We have combined X-ray crystallography of purified proteins with electron cryotomography of native arrays inside cells to reveal the arrangement of the component transmembrane receptors, histidine kinases (CheA) and CheW coupling proteins. Trimers of receptor dimers lie at the vertices of a hexagonal lattice in a "two-facing-two" configuration surrounding a ring of alternating CheA regulatory domains (P5) and CheW couplers. Whereas the CheA kinase domains (P4) project downward below the ring, the CheA dimerization domains (P3) link neighboring rings to form an extended, stable array. This highly interconnected protein architecture underlies the remarkable sensitivity and cooperative nature of transmembrane signaling in bacterial chemotaxis. |
BibTeX:
@article{Briegel2012,
author = {Briegel, Ariane and Li, Xiaoxiao and Bilwes, Alexandrine M. and Hughes, Kelly T. and Jensen, Grant J. and Crane, Brian R.},
title = {Bacterial chemoreceptor arrays are hexagonally packed trimers of receptor dimers networked by rings of kinase and coupling proteins.},
journal = {Proc Natl Acad Sci U S A},
school = {Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA.},
year = {2012},
volume = {109},
number = {10},
pages = {3766--3771},
url = {http://dx.doi.org/10.1073/pnas.1115719109},
doi = {10.1073/pnas.1115719109}
}
|
| Briegel A, Ortega DR, Huang AN, Oikonomou CM, Gunsalus RP and Jensen GJ (2015), "Structural conservation of chemotaxis machinery across Archaea and Bacteria.", Environ Microbiol Rep. Vol. 7(3), pp. 414-419. |
| Abstract: Chemotaxis allows cells to sense and respond to their environment. In Bacteria, stimuli are detected by arrays of chemoreceptors that relay the signal to a two-component regulatory system. These arrays take the form of highly stereotyped super-lattices comprising hexagonally packed trimers-of-receptor-dimers networked by rings of histidine kinase and coupling proteins. This structure is conserved across chemotactic Bacteria, and between membrane-bound and cytoplasmic arrays, and gives rise to the highly cooperative, dynamic nature of the signalling system. The chemotaxis system, absent in eukaryotes, is also found in Archaea, where its structural details remain uncharacterized. Here we provide evidence that the chemotaxis machinery was not present in the last archaeal common ancestor, but rather was introduced in one of the waves of lateral gene transfer that occurred after the branching of Eukaryota but before the diversification of Euryarchaeota. Unlike in Bacteria, the chemotaxis system then evolved largely vertically in Archaea, with very few subsequent successful lateral gene transfer events. By electron cryotomography, we find that the structure of both membrane-bound and cytoplasmic chemoreceptor arrays is conserved between Bacteria and Archaea, suggesting the fundamental importance of this signalling architecture across diverse prokaryotic lifestyles. |
BibTeX:
@article{Briegel2015,
author = {Briegel, Ariane and Ortega, Davi R. and Huang, Audrey N. and Oikonomou, Catherine M. and Gunsalus, Robert P. and Jensen, Grant J.},
title = {Structural conservation of chemotaxis machinery across Archaea and Bacteria.},
journal = {Environ Microbiol Rep},
school = {Howard Hughes Medical Institute, 1200 E. California Blvd., Pasadena, CA, 91125, USA.},
year = {2015},
volume = {7},
number = {3},
pages = {414--419},
url = {http://dx.doi.org/10.1111/1758-2229.12265},
doi = {10.1111/1758-2229.12265}
}
|
| Briegel A, Ortega DR, Mann P, Kjær A, Ringgaard S and Jensen GJ (2016), "Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 113, pp. 10412-10417. |
| Abstract: Nearly all motile bacterial cells use a highly sensitive and adaptable sensory system to detect changes in nutrient concentrations in the environment and guide their movements toward attractants and away from repellents. The best-studied bacterial chemoreceptor arrays are membrane-bound. Many motile bacteria contain one or more additional, sometimes purely cytoplasmic, chemoreceptor systems. Vibrio cholerae contains three chemotaxis clusters (I, II, and III). Here, using electron cryotomography, we explore V. cholerae's cytoplasmic chemoreceptor array and establish that it is formed by proteins from cluster I. We further identify a chemoreceptor with an unusual domain architecture, DosM, which is essential for formation of the cytoplasmic arrays. DosM contains two signaling domains and spans the two-layered cytoplasmic arrays. Finally, we present evidence suggesting that this type of receptor is important for the structural stability of the cytoplasmic array. |
BibTeX:
@article{Briegel2016,
author = {Briegel, Ariane and Ortega, Davi R and Mann, Petra and Kjær, Andreas and Ringgaard, Simon and Jensen, Grant J},
title = {Chemotaxis cluster 1 proteins form cytoplasmic arrays in Vibrio cholerae and are stabilized by a double signaling domain receptor DosM.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2016},
volume = {113},
pages = {10412--10417},
doi = {10.1073/pnas.1604693113}
}
|
| Briegel A, Pilhofer M, Mastronarde DN and Jensen GJ (2013), "The challenge of determining handedness in electron tomography and the use of DNA origami gold nanoparticle helices as molecular standards.", Journal of structural biology. Vol. 183, pp. 95-98. |
| Abstract: The apparent handedness of an EM-tomography reconstruction depends on a number of conventions and can be confused in many ways. As the number of different hardware and software combinations being used for electron tomography continue to climb, and the reconstructions being produced reach higher and higher resolutions, the need to verify the hand of the results has increased. Here we enumerate various steps in a typical tomography experiment that affect handedness and show that DNA origami gold nanoparticle helices can be used as convenient and fail-safe handedness standards. |
BibTeX:
@article{Briegel2013a,
author = {Briegel, Ariane and Pilhofer, Martin and Mastronarde, David N and Jensen, Grant J},
title = {The challenge of determining handedness in electron tomography and the use of DNA origami gold nanoparticle helices as molecular standards.},
journal = {Journal of structural biology},
year = {2013},
volume = {183},
pages = {95--98},
doi = {10.1016/j.jsb.2013.04.008}
}
|
| Briegel A, Wong ML, Hodges HL, Oikonomou CM, Piasta KN, Harris MJ, Fowler DJ, Thompson LK, Falke JJ, Kiessling LL and Jensen GJ (2014), "New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography.", Biochemistry. Vol. 53(10), pp. 1575-1585. |
| Abstract: Bacterial chemoreceptors cluster in highly ordered, cooperative, extended arrays with a conserved architecture, but the principles that govern array assembly remain unclear. Here we show images of cellular arrays as well as selected chemoreceptor complexes reconstituted in vitro that reveal new principles of array structure and assembly. First, in every case, receptors clustered in a trimers-of-dimers configuration, suggesting this is a highly favored fundamental building block. Second, these trimers-of-receptor dimers exhibited great versatility in the kinds of contacts they formed with each other and with other components of the signaling pathway, although only one architectural type occurred in native arrays. Third, the membrane, while it likely accelerates the formation of arrays, was neither necessary nor sufficient for lattice formation. Molecular crowding substituted for the stabilizing effect of the membrane and allowed cytoplasmic receptor fragments to form sandwiched lattices that strongly resemble the cytoplasmic chemoreceptor arrays found in some bacterial species. Finally, the effective determinant of array structure seemed to be CheA and CheW, which formed a "superlattice" of alternating CheA-filled and CheA-empty rings that linked receptor trimers-of-dimer units into their native hexagonal lattice. While concomitant overexpression of receptors, CheA, and CheW yielded arrays with native spacing, the CheA occupancy was lower and less ordered, suggesting that temporal and spatial coordination of gene expression driven by a single transcription factor may be vital for full order, or that array overgrowth may trigger a disassembly process. The results described here provide new insights into the assembly intermediates and assembly mechanism of this massive macromolecular complex. |
BibTeX:
@article{Briegel2014,
author = {Briegel, Ariane and Wong, Margaret L. and Hodges, Heather L. and Oikonomou, Catherine M. and Piasta, Kene N. and Harris, Michael J. and Fowler, Daniel J. and Thompson, Lynmarie K. and Falke, Joseph J. and Kiessling, Laura L. and Jensen, Grant J.},
title = {New insights into bacterial chemoreceptor array structure and assembly from electron cryotomography.},
journal = {Biochemistry},
school = {Division of Biology, California Institute of Technology , 1200 East California Boulevard, Pasadena, California 91125, United States.},
year = {2014},
volume = {53},
number = {10},
pages = {1575--1585},
url = {http://dx.doi.org/10.1021/bi5000614},
doi = {10.1021/bi5000614}
}
|
| Brown JR, Schwartz CL, Heumann JM, Dawson SC and Hoenger A (2016), "A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.", J Struct Biol. Vol. 194(1), pp. 38-48. |
| Abstract: Giardia lamblia is a protistan parasite that infects and colonizes the small intestine of mammals. It is widespread and particularly endemic in the developing world. Here we present a detailed structural study by 3-D negative staining and cryo-electron tomography of a unique Giardia organelle, the ventral disc. The disc is composed of a regular array of microtubules and associated sheets, called microribbons that form a large spiral, held together by a myriad of mostly unknown associated proteins. In a previous study we analyzed by cryo-electron tomography the central microtubule portion (here called disc body) of the ventral disc and found a large portion of microtubule associated inner (MIPs) and outer proteins (MAPs) that render these microtubules hyper-stable. With this follow-up study we expanded our 3-D analysis to different parts of the disc such as the ventral and dorsal areas of the overlap zone, as well as the outer disc margin. There are intrinsic location-specific characteristics in the composition of microtubule-associated proteins between these regions, as well as large differences between the overall architecture of microtubules and microribbons. The lateral packing of microtubule-microribbon complexes varies substantially, and closer packing often comes with contracted lateral tethers that seem to hold the disc together. It appears that the marginal microtubule-microribbon complexes function as outer, laterally contractible lids that may help the cell to clamp onto the intestinal microvilli. Furthermore, we analyzed length, quantity, curvature and distribution between different zones of the disc, which we found to differ from previous publications. |
BibTeX:
@article{Brown2016,
author = {Brown, Joanna R. and Schwartz, Cindi L. and Heumann, John M. and Dawson, Scott C. and Hoenger, Andreas},
title = {A detailed look at the cytoskeletal architecture of the Giardia lamblia ventral disc.},
journal = {J Struct Biol},
school = {University of Colorado, Dept. MCD Biology, Boulder, CO 80309, USA. Electronic address: Hoenger@colorado.edu.},
year = {2016},
volume = {194},
number = {1},
pages = {38--48},
url = {http://dx.doi.org/10.1016/j.jsb.2016.01.011},
doi = {10.1016/j.jsb.2016.01.011}
}
|
| Bui KH, Pigino G and Ishikawa T (2011), "Three-dimensional structural analysis of eukaryotic flagella/cilia by electron cryo-tomography.", J Synchrotron Radiat. Vol. 18(1), pp. 2-5. |
| Abstract: Electron cryo-tomography is a potential approach to analyzing the three-dimensional conformation of frozen hydrated biological macromolecules using electron microscopy. Since projections of each individual object illuminated from different orientations are merged, electron tomography is capable of structural analysis of such heterogeneous environments as in vivo or with polymorphism, although radiation damage and the missing wedge are severe problems. Here, recent results on the structure of eukaryotic flagella, which is an ATP-driven bending organelle, from green algae Chlamydomonas are presented. Tomographic analysis reveals asymmetric molecular arrangements, especially that of the dynein motor proteins, in flagella, giving insight into the mechanism of planar asymmetric bending motion. Methodological challenges to obtaining higher-resolution structures from this technique are also discussed. |
BibTeX:
@article{Bui2011,
author = {Bui, Khanh Huy and Pigino, Gaia and Ishikawa, Takashi},
title = {Three-dimensional structural analysis of eukaryotic flagella/cilia by electron cryo-tomography.},
journal = {J Synchrotron Radiat},
school = {Paul Scherrer Institute, 5232 Villigen PSI, Switzerland.},
year = {2011},
volume = {18},
number = {1},
pages = {2--5},
url = {http://dx.doi.org/10.1107/S0909049510036812},
doi = {10.1107/S0909049510036812}
}
|
| Burgoyne T, Heumann JM, Morris EP, Knupp C, Liu J, Reedy MK, Taylor KA, Wang K and Luther PK (2019), "Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 116, pp. 15534-15539. |
| Abstract: Striated muscle enables movement in all animals by the contraction of myriads of sarcomeres joined end to end by the Z-bands. The contraction is due to tension generated in each sarcomere between overlapping arrays of actin and myosin filaments. At the Z-band, actin filaments from adjoining sarcomeres overlap and are cross-linked in a regular pattern mainly by the protein α-actinin. The Z-band is dynamic, reflected by the 2 regular patterns seen in transverse section electron micrographs; the so-called small-square and basketweave forms. Although these forms are attributed, respectively, to relaxed and actively contracting muscles, the basketweave form occurs in certain relaxed muscles as in the muscle studied here. We used electron tomography and subtomogram averaging to derive the 3D structure of the Z-band in the swimbladder sonic muscle of type I male plainfin midshipman fish ( , into which we docked the crystallographic structures of actin and α-actinin. The α-actinin links run diagonally between connected pairs of antiparallel actin filaments and are oriented at an angle of about 25° away from the actin filament axes. The slightly curved and flattened structure of the α-actinin rod has a distinct fit into the map. The Z-band model provides a detailed understanding of the role of α-actinin in transmitting tension between actin filaments in adjoining sarcomeres. |
BibTeX:
@article{Burgoyne2019,
author = {Burgoyne, Thomas and Heumann, John M. and Morris, Edward P. and Knupp, Carlo and Liu, Jun and Reedy, Michael K. and Taylor, Kenneth A. and Wang, Kuan and Luther, Pradeep K.},
title = {Three-dimensional structure of the basketweave Z-band in midshipman fish sonic muscle.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2019},
volume = {116},
pages = {15534--15539},
doi = {10.1073/pnas.1902235116}
}
|
| Burgoyne T, Morris EP and Luther PK (2015), "Three-Dimensional Structure of Vertebrate Muscle Z-Band: The Small-Square Lattice Z-Band in Rat Cardiac Muscle.", J Mol Biol. Vol. 427(22), pp. 3527-3537. |
| Abstract: The Z-band in vertebrate striated muscle crosslinks actin filaments of opposite polarity from adjoining sarcomeres and transmits tension along myofibrils during muscular contraction. It is also the location of a number of proteins involved in signalling and myofibrillogenesis; mutations in these proteins lead to myopathies. Understanding the high-resolution structure of the Z-band will help us understand its role in muscle contraction and the role of these proteins in the function of muscle. The appearance of the Z-band in transverse-section electron micrographs typically resembles a small-square lattice or a basketweave appearance. In longitudinal sections, the Z-band width varies more with muscle type than species: slow skeletal and cardiac muscles have wider Z-bands than fast skeletal muscles. As the Z-band is periodic, Fourier methods have previously been used for three-dimensional structural analysis. To cope with variations in the periodic structure of the Z-band, we have used subtomogram averaging of tomograms of rat cardiac muscle in which subtomograms are extracted and compared and similar ones are averaged. We show that the Z-band comprises four to six layers of links, presumably α-actinin, linking antiparallel overlapping ends of the actin filaments from the adjoining sarcomeres. The reconstruction shows that the terminal 5-7nm of the actin filaments within the Z-band is devoid of any α-actinin links and is likely to be the location of capping protein CapZ. |
BibTeX:
@article{Burgoyne2015,
author = {Burgoyne, Thomas and Morris, Edward P. and Luther, Pradeep K.},
title = {Three-Dimensional Structure of Vertebrate Muscle Z-Band: The Small-Square Lattice Z-Band in Rat Cardiac Muscle.},
journal = {J Mol Biol},
school = {Imperial College London, London SW7 2AZ, United Kingdom. Electronic address: p.luther@imperial.ac.uk.},
year = {2015},
volume = {427},
number = {22},
pages = {3527--3537},
url = {http://dx.doi.org/10.1016/j.jmb.2015.08.018},
doi = {10.1016/j.jmb.2015.08.018}
}
|
| Burns JE and Pachl JK (1989), "Uniform self-stabilizing rings", In ACM Trans. Program. Lang. Syst.., pp. 330–344. Association for Computing Machinery. |
| Abstract: A self-stabilizing system has the property that, no matter how it is perturbed, it eventually returns to a legitimate configuration. Dijkstra originally introduced the self-stabilization problem and gave several solutions for a ring of processors in his 1974 Communications of the ACM paper. His solutions use a distinguished processor in the ring, which effectively acts as a controlling element to drive the system toward stability. Dijkstra has observed that a distinguished processor is essential if the number of processors in the ring is composite. We show, by presenting a protocol and proving its correctness, that there is a self-stabilizing system with no distinguished processor if the size of the ring is prime. The basic protocol uses Θ (n2) states in each processor when n is the size of the ring. We modify the basic protocol to obtain one that uses Θ (n2/ln n) states. |
BibTeX:
@article{Burns1989,
author = {J. E. Burns and Jan K. Pachl},
title = {Uniform self-stabilizing rings},
booktitle = {ACM Trans. Program. Lang. Syst.},
publisher = {Association for Computing Machinery},
year = {1989},
pages = {330–344},
url = {https://doi.org/10.1145/63264.63403},
doi = {10.1145/63264.63403}
}
|
| Cai S, Böck D, Pilhofer M and Gan L (2018), "The in situ structures of mono-, di-, and trinucleosomes in human heterochromatin.", Molecular biology of the cell. Vol. 29, pp. 2450-2457. |
| Abstract: The in situ three-dimensional organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level. |
BibTeX:
@article{Cai2018,
author = {Cai, Shujun and Böck, Désirée and Pilhofer, Martin and Gan, Lu},
title = {The in situ structures of mono-, di-, and trinucleosomes in human heterochromatin.},
journal = {Molecular biology of the cell},
year = {2018},
volume = {29},
pages = {2450--2457},
doi = {10.1091/mbc.E18-05-0331}
}
|
| Cai S, Chen C, Tan ZY, Huang Y, Shi J and Gan L (2018), "Cryo-ET reveals the macromolecular reorganization of , javax.xml.bind.JAXBElement@58aed146, mitotic chromosomes in vivo.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 115, pp. 10977-10982. |
| Abstract: Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription. Here, we use cryo-electron tomography (cryo-ET) to determine the 3D arrangement of nuclear macromolecular complexes, including nucleosomes, in frozen-hydrated cells. Using 3D classification analysis, we did not find evidence that nucleosomes resembling the crystal structure are abundant. This observation and those from other groups support the notion that a subset of fission yeast nucleosomes may be partially unwrapped in vivo. In both interphase and mitotic cells, there is also no evidence of monolithic structures the size of Hi-C domains. The chromatin is mingled with two features: pockets, which are positions free of macromolecular complexes; and "megacomplexes," which are multimegadalton globular complexes like preribosomes. Mitotic chromatin is more crowded than interphase chromatin in subtle ways. Nearest-neighbor distance analyses show that mitotic chromatin is more compacted at the oligonucleosome than the dinucleosome level. Like interphase, mitotic chromosomes contain megacomplexes and pockets. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: a subset of genes is up-regulated. |
BibTeX:
@article{Cai2018a,
author = {Cai, Shujun and Chen, Chen and Tan, Zhi Yang and Huang, Yinyi and Shi, Jian and Gan, Lu},
title = {Cryo-ET reveals the macromolecular reorganization of , javax.xml.bind.JAXBElement@58aed146, mitotic chromosomes in vivo.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2018},
volume = {115},
pages = {10977--10982},
doi = {10.1073/pnas.1720476115}
}
|
| Cai S, Song Y, Chen C, Shi J and Gan L (2018), "Natural chromatin is heterogeneous and self-associates in vitro", Molecular biology of the cell. Vol. 29(13), pp. 1652-1663. Am Soc Cell Biol.
[BibTeX] |
BibTeX:
@article{Cai2018b,
author = {Cai, Shujun and Song, Yajiao and Chen, Chen and Shi, Jian and Gan, Lu},
title = {Natural chromatin is heterogeneous and self-associates in vitro},
journal = {Molecular biology of the cell},
publisher = {Am Soc Cell Biol},
year = {2018},
volume = {29},
number = {13},
pages = {1652--1663}
}
|
| Cai S, Tan ZY, Nie X, Shi J and Gan L (2020), "Macromolecular and biochemical changes of G0 fission yeast nuclei", bioRxiv. Cold Spring Harbor Laboratory.
[BibTeX] |
BibTeX:
@article{Cai2020,
author = {Cai, Shujun and Tan, Zhi Yang and Nie, Xin and Shi, Jian and Gan, Lu},
title = {Macromolecular and biochemical changes of G0 fission yeast nuclei},
journal = {bioRxiv},
publisher = {Cold Spring Harbor Laboratory},
year = {2020}
}
|
| Carbajal-González BI, Heuser T, Fu X, Lin J, Smith BW, Mitchell DR and Nicastro D (2013), "Conserved structural motifs in the central pair complex of eukaryotic flagella.", Cytoskeleton (Hoboken). Vol. 70(2), pp. 101-120. |
| Abstract: Cilia and flagella are conserved hair-like appendages of eukaryotic cells that function as sensing and motility generating organelles. Motility is driven by thousands of axonemal dyneins that require precise regulation. One essential motility regulator is the central pair complex (CPC) and many CPC defects cause paralysis of cilia/flagella. Several human diseases, such as immotile cilia syndrome, show CPC abnormalities, but little is known about the detailed three-dimensional (3D) structure and function of the CPC. The CPC is located in the center of typical [9+2] cilia/flagella and is composed of two singlet microtubules (MTs), each with a set of associated projections that extend toward the surrounding nine doublet MTs. Using cryo-electron tomography coupled with subtomogram averaging, we visualized and compared the 3D structures of the CPC in both the green alga Chlamydomonas and the sea urchin Strongylocentrotus at the highest resolution published to date. Despite the evolutionary distance between these species, their CPCs exhibit remarkable structural conservation. We identified several new projections, including those that form the elusive sheath, and show that the bridge has a more complex architecture than previously thought. Organism-specific differences include the presence of MT inner proteins in Chlamydomonas, but not Strongylocentrotus, and different overall outlines of the highly connected projection network, which forms a round-shaped cylinder in algae, but is more oval in sea urchin. These differences could be adaptations to the mechanical requirements of the rotating CPC in Chlamydomonas, compared to the Strongylocentrotus CPC which has a fixed orientation. |
BibTeX:
@article{Carbajal-Gonzalez2013,
author = {Carbajal-González, Blanca I. and Heuser, Thomas and Fu, Xiaofeng and Lin, Jianfeng and Smith, Brandon W. and Mitchell, David R. and Nicastro, Daniela},
title = {Conserved structural motifs in the central pair complex of eukaryotic flagella.},
journal = {Cytoskeleton (Hoboken)},
school = {Department of Biology, Rosenstiel Center, MS029, Brandeis University, 415 South Street, Waltham, MA 02454, USA.},
year = {2013},
volume = {70},
number = {2},
pages = {101--120},
url = {http://dx.doi.org/10.1002/cm.21094},
doi = {10.1002/cm.21094}
}
|
| Chang Y-W, Kjær A, Ortega DR, Kovacikova G, Sutherland JA, Rettberg LA, Taylor RK and Jensen GJ (2017), "Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography.", Nature microbiology. Vol. 2, pp. 16269. |
| Abstract: Type IV pili (T4P) are filamentous appendages found on many Bacteria and Archaea. They are helical fibres of pilin proteins assembled by a multi-component macromolecular machine we call the basal body. Based on pilin features, T4P are classified into type IVa pili (T4aP) and type IVb pili (T4bP)1,2. T4aP are more widespread and are involved in cell motility3, DNA transfer4, host predation5 and electron transfer6. T4bP are less prevalent and are mainly found in enteropathogenic bacteria, where they play key roles in host colonization7. Following similar work on T4aP machines8,9, here we use electron cryotomography10 to reveal the three-dimensional in situ structure of a T4bP machine in its piliated and non-piliated states. The specific machine we analyse is the Vibrio cholerae toxin-coregulated pilus machine (TCPM). Although only about half of the components of the TCPM show sequence homology to components of the previously analysed Myxococcus xanthus T4aP machine (T4aPM), we find that their structures are nevertheless remarkably similar. Based on homologies with components of the M. xanthus T4aPM and additional reconstructions of TCPM mutants in which the non-homologous proteins are individually deleted, we propose locations for all eight TCPM components within the complex. Non-homologous proteins in the T4aPM and TCPM are found to form similar structures, suggesting new hypotheses for their functions and evolutionary histories. |
BibTeX:
@article{Chang2017b,
author = {Chang, Yi-Wei and Kjær, Andreas and Ortega, Davi R and Kovacikova, Gabriela and Sutherland, John A and Rettberg, Lee A and Taylor, Ronald K and Jensen, Grant J},
title = {Architecture of the Vibrio cholerae toxin-coregulated pilus machine revealed by electron cryotomography.},
journal = {Nature microbiology},
year = {2017},
volume = {2},
pages = {16269},
doi = {10.1038/nmicrobiol.2016.269}
}
|
| Chang Y-W, Rettberg LA, Ortega DR and Jensen GJ (2017), "In vivo structures of an intact type VI secretion system revealed by electron cryotomography.", EMBO reports. Vol. 18, pp. 1090-1099. |
| Abstract: The type VI secretion system (T6SS) is a versatile molecular weapon used by many bacteria against eukaryotic hosts or prokaryotic competitors. It consists of a cytoplasmic bacteriophage tail-like structure anchored in the bacterial cell envelope via a cytoplasmic baseplate and a periplasmic membrane complex. Rapid contraction of the sheath in the bacteriophage tail-like structure propels an inner tube/spike complex through the target cell envelope to deliver effectors. While structures of purified contracted sheath and purified membrane complex have been solved, because sheaths contract upon cell lysis and purification, no structure is available for the extended sheath. Structural information about the baseplate is also lacking. Here, we use electron cryotomography to directly visualize intact T6SS structures inside Myxococcus xanthus cells. Using sub-tomogram averaging, we resolve the structure of the extended sheath and membrane-associated components including the baseplate. Moreover, we identify novel extracellular bacteriophage tail fiber-like antennae. These results provide new structural insights into how the extended sheath prevents premature disassembly and how this sophisticated machine may recognize targets. |
BibTeX:
@article{Chang2017,
author = {Chang, Yi-Wei and Rettberg, Lee A and Ortega, Davi R and Jensen, Grant J},
title = {In vivo structures of an intact type VI secretion system revealed by electron cryotomography.},
journal = {EMBO reports},
year = {2017},
volume = {18},
pages = {1090--1099},
doi = {10.15252/embr.201744072}
}
|
| Chen C, Lim HH, Shi J, Tamura S, Maeshima K, Surana U and Gan L (2016), "Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo.", Molecular biology of the cell. Vol. 27, pp. 3357-3368. |
| Abstract: Chromatin organization has an important role in the regulation of eukaryotic systems. Although recent studies have refined the three-dimensional models of chromatin organization with high resolution at the genome sequence level, little is known about how the most fundamental units of chromatin-nucleosomes-are positioned in three dimensions in vivo. Here we use electron cryotomography to study chromatin organization in the budding yeast Saccharomyces cerevisiae Direct visualization of yeast nuclear densities shows no evidence of 30-nm fibers. Aside from preribosomes and spindle microtubules, few nuclear structures are larger than a tetranucleosome. Yeast chromatin does not form compact structures in interphase or mitosis and is consistent with being in an "open" configuration that is conducive to high levels of transcription. From our study and those of others, we propose that yeast can regulate its transcription using local nucleosome-nucleosome associations. |
BibTeX:
@article{Chen2016,
author = {Chen, Chen and Lim, Hong Hwa and Shi, Jian and Tamura, Sachiko and Maeshima, Kazuhiro and Surana, Uttam and Gan, Lu},
title = {Budding yeast chromatin is dispersed in a crowded nucleoplasm in vivo.},
journal = {Molecular biology of the cell},
year = {2016},
volume = {27},
pages = {3357--3368},
doi = {10.1091/mbc.E16-07-0506}
}
|
| Chen S, Beeby M, Murphy GE, Leadbetter JR, Hendrixson DR, Briegel A, Li Z, Shi J, Tocheva EI, Müller A, Dobro MJ and Jensen GJ (2011), "Structural diversity of bacterial flagellar motors.", EMBO J. Vol. 30(14), pp. 2972-2981. |
| Abstract: The bacterial flagellum is one of nature's most amazing and well-studied nanomachines. Its cell-wall-anchored motor uses chemical energy to rotate a microns-long filament and propel the bacterium towards nutrients and away from toxins. While much is known about flagellar motors from certain model organisms, their diversity across the bacterial kingdom is less well characterized, allowing the occasional misrepresentation of the motor as an invariant, ideal machine. Here, we present an electron cryotomographical survey of flagellar motor architectures throughout the Bacteria. While a conserved structural core was observed in all 11 bacteria imaged, surprisingly novel and divergent structures as well as different symmetries were observed surrounding the core. Correlating the motor structures with the presence and absence of particular motor genes in each organism suggested the locations of five proteins involved in the export apparatus including FliI, whose position below the C-ring was confirmed by imaging a deletion strain. The combination of conserved and specially-adapted structures seen here sheds light on how this complex protein nanomachine has evolved to meet the needs of different species. |
BibTeX:
@article{Chen2011,
author = {Chen, Songye and Beeby, Morgan and Murphy, Gavin E. and Leadbetter, Jared R. and Hendrixson, David R. and Briegel, Ariane and Li, Zhuo and Shi, Jian and Tocheva, Elitza I. and Müller, Axel and Dobro, Megan J. and Jensen, Grant J.},
title = {Structural diversity of bacterial flagellar motors.},
journal = {EMBO J},
school = {Division of Biology, California Institute of Technology, Pasadena, CA, USA.},
year = {2011},
volume = {30},
number = {14},
pages = {2972--2981},
url = {http://dx.doi.org/10.1038/emboj.2011.186},
doi = {10.1038/emboj.2011.186}
}
|
| Ciesielski PN, Matthews JF, Tucker MP, Beckham GT, Crowley MF, Himmel ME and Donohoe BS (2013), "3D electron tomography of pretreated biomass informs atomic modeling of cellulose microfibrils.", ACS Nano. Vol. 7(9), pp. 8011-8019. |
| Abstract: Fundamental insights into the macromolecular architecture of plant cell walls will elucidate new structure-property relationships and facilitate optimization of catalytic processes that produce fuels and chemicals from biomass. Here we introduce computational methodology to extract nanoscale geometry of cellulose microfibrils within thermochemically treated biomass directly from electron tomographic data sets. We quantitatively compare the cell wall nanostructure in corn stover following two leading pretreatment strategies: dilute acid with iron sulfate co-catalyst and ammonia fiber expansion (AFEX). Computational analysis of the tomographic data is used to extract mathematical descriptions for longitudinal axes of cellulose microfibrils from which we calculate their nanoscale curvature. These nanostructural measurements are used to inform the construction of atomistic models that exhibit features of cellulose within real, process-relevant biomass. By computational evaluation of these atomic models, we propose relationships between the crystal structure of cellulose Iβ and the nanoscale geometry of cellulose microfibrils. |
BibTeX:
@article{Ciesielski2013,
author = {Ciesielski, Peter N. and Matthews, James F. and Tucker, Melvin P. and Beckham, Gregg T. and Crowley, Michael F. and Himmel, Michael E. and Donohoe, Bryon S.},
title = {3D electron tomography of pretreated biomass informs atomic modeling of cellulose microfibrils.},
journal = {ACS Nano},
school = {Biosciences Center, National Renewable Energy Laboratory , 15013 Denver West Parkway, Golden, Colorado 80401, United States.},
year = {2013},
volume = {7},
number = {9},
pages = {8011--8019},
url = {http://dx.doi.org/10.1021/nn4031542},
doi = {10.1021/nn4031542}
}
|
| Cope J, Gilbert S, Rayment I, Mastronarde D and Hoenger A (2010), "Cryo-electron tomography of microtubule-kinesin motor complexes.", J Struct Biol. Vol. 170(2), pp. 257-265. |
| Abstract: Microtubules complexed with molecular motors of the kinesin family or non-motor microtubule associated proteins (MAPs) such as tau or EB1 have been the subject of cryo-electron microcopy based 3-D studies for several years. Most of these studies that targeted complexes with intact microtubules have been carried out by helical 3-D reconstruction, while few were analyzed by single particle approaches or from 2-D crystalline arrays. Helical reconstruction of microtubule-MAP or motor complexes has been extremely successful but by definition, all helical 3-D reconstruction attempts require perfectly helical assemblies, which presents a serious limitation and confines the attempts to 15- or 16-protofilament microtubules, microtubule configurations that are very rare in nature. The rise of cryo-electron tomography within the last few years has now opened a new avenue towards solving 3-D structures of microtubule-MAP complexes that do not form helical assemblies, most importantly for the subject here, all microtubules that exhibit a lattice seam. In addition, not all motor domains or MAPs decorate the microtubule surface regularly enough to match the underlying microtubule lattice, or they adopt conformations that deviate from helical symmetry. Here we demonstrate the power and limitation of cryo-electron tomography using two kinesin motor domains, the monomeric Eg5 motor domain, and the heterodimeric Kar3Vik1 motor. We show here that tomography does not exclude the possibility of post-tomographic averaging when identical sub-volumes can be extracted from tomograms and in both cases we were able to reconstruct 3-D maps of conformations that are not possible to obtain using helical or other averaging-based methods. |
BibTeX:
@article{Cope2010,
author = {Cope, Julia and Gilbert, Susan and Rayment, Ivan and Mastronarde, David and Hoenger, Andreas},
title = {Cryo-electron tomography of microtubule-kinesin motor complexes.},
journal = {J Struct Biol},
school = {The Boulder Laboratory for 3-D Microscopy of Cells, University of Colorado at Boulder, Department of Molecular, Cellular, and Developmental Biology, Boulder, CO 80309-0347, USA.},
year = {2010},
volume = {170},
number = {2},
pages = {257--265},
url = {http://dx.doi.org/10.1016/j.jsb.2009.12.004},
doi = {10.1016/j.jsb.2009.12.004}
}
|
| Cope J, Heumann J and Hoenger A (2011), "Cryo-electron tomography for structural characterization of macromolecular complexes.", Curr Protoc Protein Sci. Vol. Chapter 17, pp. Unit17.13. |
| Abstract: Cryo-electron tomography (cryo-ET) is an emerging 3-D reconstruction technology that combines the principles of tomographic 3-D reconstruction with the unmatched structural preservation of biological matter embedded in vitreous ice. Cryo-ET is particularly suited to investigating cell-biological samples and large macromolecular structures that are too polymorphic to be reconstructed by classical averaging-based 3-D reconstruction procedures. This unit aims to make cryo-ET accessible to newcomers and discusses the specialized equipment required, as well as relevant advantages and hurdles associated with sample preparation by vitrification and cryo-ET. Protocols describe specimen preparation, data recording and 3-D data reconstruction for cryo-ET, with a special focus on macromolecular complexes. A step-by-step procedure for specimen vitrification by plunge freezing is provided, followed by the general practicalities of tilt-series acquisition for cryo-ET, including advice on how to select an area appropriate for acquiring a tilt series. A brief introduction to the underlying computational reconstruction principles applied in tomography is described, along with instructions for reconstructing a tomogram from cryo-tilt series data. Finally, a method is detailed for extracting small subvolumes containing identical macromolecular structures from tomograms for alignment and averaging as a means to increase the signal-to-noise ratio and eliminate missing wedge effects inherent in tomographic reconstructions. |
BibTeX:
@article{Cope2011,
author = {Cope, Julia and Heumann, John and Hoenger, Andreas},
title = {Cryo-electron tomography for structural characterization of macromolecular complexes.},
journal = {Curr Protoc Protein Sci},
school = {Department of Molecular, Cellular and Developmental Biology, University of Colorado Boulder, Boulder, Colorado, USA.},
year = {2011},
volume = {Chapter 17},
pages = {Unit17.13},
url = {http://dx.doi.org/10.1002/0471140864.ps1713s65},
doi = {10.1002/0471140864.ps1713s65}
}
|
| Dahan I, Sorrentino S, Boujemaa-Paterski R and Medalia O (2018), "Tiopronin-Protected Gold Nanoparticles as a Potential Marker for Cryo-EM and Tomography", Structure. Vol. 26(10), pp. 1408-1413. Elsevier.
[BibTeX] |
BibTeX:
@article{Dahan2018,
author = {Dahan, Idit and Sorrentino, Simona and Boujemaa-Paterski, Rajaa and Medalia, Ohad},
title = {Tiopronin-Protected Gold Nanoparticles as a Potential Marker for Cryo-EM and Tomography},
journal = {Structure},
publisher = {Elsevier},
year = {2018},
volume = {26},
number = {10},
pages = {1408--1413}
}
|
| Daum B, Auerswald A, Gruber T, Hause G, Balbach J, Kühlbrandt W and Meister A (2016), "Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane.", Journal of structural biology. Vol. 194, pp. 375-382. |
| Abstract: The 30kDa N-BAR domain of the human Bin1 protein is essential for the generation of skeletal muscle T-tubules. By electron cryo-microscopy and electron cryo-tomography with a direct electron detector, we found that Bin1-N-BAR domains assemble into scaffolds of low long-range order that form flexible membrane tubules. The diameter of the tubules closely matches the curved shape of the N-BAR domain, which depends on the composition of the target membrane. These insights are fundamental to our understanding of T-tubule formation and function in human skeletal muscle. |
BibTeX:
@article{Daum2016,
author = {Daum, Bertram and Auerswald, Andrea and Gruber, Tobias and Hause, Gerd and Balbach, Jochen and Kühlbrandt, Werner and Meister, Annette},
title = {Supramolecular organization of the human N-BAR domain in shaping the sarcolemma membrane.},
journal = {Journal of structural biology},
year = {2016},
volume = {194},
pages = {375--382},
doi = {10.1016/j.jsb.2016.03.017}
}
|
| Daum B, Vonck J, Bellack A, Chaudhury P, Reichelt R, Albers S-V, Rachel R and Kühlbrandt W (2017), "Structure and in situ organisation of the Pyrococcus furiosus archaellum machinery.", eLife. Vol. 6 |
| Abstract: The archaellum is the macromolecular machinery that Archaea use for propulsion or surface adhesion, enabling them to proliferate and invade new territories. The molecular composition of the archaellum and of the motor that drives it appears to be entirely distinct from that of the functionally equivalent bacterial flagellum and flagellar motor. Yet, the structure of the archaellum machinery is scarcely known. Using combined modes of electron cryo-microscopy (cryoEM), we have solved the structure of the Pyrococcus furiosus archaellum filament at 4.2 Å resolution and visualise the architecture and organisation of its motor complex in situ. This allows us to build a structural model combining the archaellum and its motor complex, paving the way to a molecular understanding of archaeal swimming motion. |
BibTeX:
@article{Daum2017,
author = {Daum, Bertram and Vonck, Janet and Bellack, Annett and Chaudhury, Paushali and Reichelt, Robert and Albers, Sonja-Verena and Rachel, Reinhard and Kühlbrandt, Werner},
title = {Structure and in situ organisation of the Pyrococcus furiosus archaellum machinery.},
journal = {eLife},
year = {2017},
volume = {6},
doi = {10.7554/eLife.27470}
}
|
| Davies KM, Anselmi C, Wittig I, Faraldo-Gómez JD and Kühlbrandt W (2012), "Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae.", Proc Natl Acad Sci U S A. Vol. 109(34), pp. 13602-13607. |
| Abstract: We used electron cryotomography of mitochondrial membranes from wild-type and mutant Saccharomyces cerevisiae to investigate the structure and organization of ATP synthase dimers in situ. Subtomogram averaging of the dimers to 3.7 nm resolution revealed a V-shaped structure of twofold symmetry, with an angle of 86° between monomers. The central and peripheral stalks are well resolved. The monomers interact within the membrane at the base of the peripheral stalks. In wild-type mitochondria ATP synthase dimers are found in rows along the highly curved cristae ridges, and appear to be crucial for membrane morphology. Strains deficient in the dimer-specific subunits e and g or the first transmembrane helix of subunit 4 lack both dimers and lamellar cristae. Instead, cristae are either absent or balloon-shaped, with ATP synthase monomers distributed randomly in the membrane. Computer simulations indicate that isolated dimers induce a plastic deformation in the lipid bilayer, which is partially relieved by their side-by-side association. We propose that the assembly of ATP synthase dimer rows is driven by the reduction in the membrane elastic energy, rather than by direct protein contacts, and that the dimer rows enable the formation of highly curved ridges in mitochondrial cristae. |
BibTeX:
@article{Davies2012,
author = {Davies, Karen M. and Anselmi, Claudio and Wittig, Ilka and Faraldo-Gómez, José D. and Kühlbrandt, Werner},
title = {Structure of the yeast F1Fo-ATP synthase dimer and its role in shaping the mitochondrial cristae.},
journal = {Proc Natl Acad Sci U S A},
school = {Department of Structural Biology, Max Planck Institute of Biophysics, Frankfurt am Main, Germany.},
year = {2012},
volume = {109},
number = {34},
pages = {13602--13607},
url = {http://dx.doi.org/10.1073/pnas.1204593109},
doi = {10.1073/pnas.1204593109}
}
|
| Davies KM, Blum TB and Kühlbrandt W (2018), "Conserved in situ arrangement of complex I and III, javax.xml.bind.JAXBElement@26f229e7, in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 115, pp. 3024-3029. |
| Abstract: We used electron cryo-tomography and subtomogram averaging to investigate the structure of complex I and its supramolecular assemblies in the inner mitochondrial membrane of mammals, fungi, and plants. Tomographic volumes containing complex I were averaged at ∼4 nm resolution. Principal component analysis indicated that ∼60% of complex I formed a supercomplex with dimeric complex III, while ∼40% were not associated with other respiratory chain complexes. The mutual arrangement of complex I and III was essentially conserved in all supercomplexes investigated. In addition, up to two copies of monomeric complex IV were associated with the complex I III assembly in bovine heart and the yeast , but their positions varied. No complex IV was detected in the respiratory supercomplex of the plant Instead, an ∼4.5-nm globular protein density was observed on the matrix side of the complex I membrane arm, which we assign to γ-carbonic anhydrase. Our results demonstrate that respiratory chain supercomplexes in situ have a conserved core of complex I and III , but otherwise their stoichiometry and structure varies. The conserved features of supercomplex assemblies indicate an important role in respiratory electron transfer. |
BibTeX:
@article{Davies2018,
author = {Davies, Karen M and Blum, Thorsten B and Kühlbrandt, Werner},
title = {Conserved in situ arrangement of complex I and III, javax.xml.bind.JAXBElement@26f229e7, in mitochondrial respiratory chain supercomplexes of mammals, yeast, and plants.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2018},
volume = {115},
pages = {3024--3029},
doi = {10.1073/pnas.1720702115}
}
|
| Deng Y, Chen Y, Zhang Y, Wang S, Zhang F and Sun F (2016), "ICON: 3D reconstruction with 'missing-information' restoration in biological electron tomography.", Journal of structural biology. Vol. 195, pp. 100-112. |
| Abstract: Electron tomography (ET) plays an important role in revealing biological structures, ranging from macromolecular to subcellular scale. Due to limited tilt angles, ET reconstruction always suffers from the 'missing wedge' artifacts, thus severely weakens the further biological interpretation. In this work, we developed an algorithm called Iterative Compressed-sensing Optimized Non-uniform fast Fourier transform reconstruction (ICON) based on the theory of compressed-sensing and the assumption of sparsity of biological specimens. ICON can significantly restore the missing information in comparison with other reconstruction algorithms. More importantly, we used the leave-one-out method to verify the validity of restored information for both simulated and experimental data. The significant improvement in sub-tomogram averaging by ICON indicates its great potential in the future application of high-resolution structural determination of macromolecules in situ. |
BibTeX:
@article{Deng2016,
author = {Deng, Yuchen and Chen, Yu and Zhang, Yan and Wang, Shengliu and Zhang, Fa and Sun, Fei},
title = {ICON: 3D reconstruction with 'missing-information' restoration in biological electron tomography.},
journal = {Journal of structural biology},
year = {2016},
volume = {195},
pages = {100--112},
doi = {10.1016/j.jsb.2016.04.004}
}
|
| Dent KC, Thompson R, Barker AM, Hiscox JA, Barr JN, Stockley PG and Ranson NA (2013), "The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release.", Structure. Vol. 21(7), pp. 1225-1234. |
| Abstract: Simple, spherical RNA viruses have well-understood, symmetric protein capsids, but little structural information is available for their asymmetric components, such as minor proteins and their genomes, which are vital for infection. Here, we report an asymmetric structure of bacteriophage MS2, attached to its receptor, the F-pilus. Cryo-electron tomography and subtomographic averaging of such complexes result in a structure containing clear density for the packaged genome, implying that the conformation of the genome is the same in each virus particle. The data also suggest that the single-copy viral maturation protein breaks the symmetry of the capsid, occupying a position that would be filled by a coat protein dimer in an icosahedral shell. This capsomere can thus fulfill its known biological roles in receptor and genome binding and suggests an exit route for the genome during infection. |
BibTeX:
@article{Dent2013,
author = {Dent, Kyle C. and Thompson, Rebecca and Barker, Amy M. and Hiscox, Julian A. and Barr, John N. and Stockley, Peter G. and Ranson, Neil A.},
title = {The asymmetric structure of an icosahedral virus bound to its receptor suggests a mechanism for genome release.},
journal = {Structure},
school = {Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.},
year = {2013},
volume = {21},
number = {7},
pages = {1225--1234},
url = {http://dx.doi.org/10.1016/j.str.2013.05.012},
doi = {10.1016/j.str.2013.05.012}
}
|
| Diebolder CA, Beurskens FJ, de Jong RN, Koning RI, Strumane K, Lindorfer MA, Voorhorst M, Ugurlar D, Rosati S, Heck AJR, van de Winkel JGJ, Wilson IA, Koster AJ, Taylor RP, Saphire EO, Burton DR, Schuurman J, Gros P and Parren PWHI (2014), "Complement is activated by IgG hexamers assembled at the cell surface.", Science. Vol. 343(6176), pp. 1260-1263. |
| Abstract: Complement activation by antibodies bound to pathogens, tumors, and self antigens is a critical feature of natural immune defense, a number of disease processes, and immunotherapies. How antibodies activate the complement cascade, however, is poorly understood. We found that specific noncovalent interactions between Fc segments of immunoglobulin G (IgG) antibodies resulted in the formation of ordered antibody hexamers after antigen binding on cells. These hexamers recruited and activated C1, the first component of complement, thereby triggering the complement cascade. The interactions between neighboring Fc segments could be manipulated to block, reconstitute, and enhance complement activation and killing of target cells, using all four human IgG subclasses. We offer a general model for understanding antibody-mediated complement activation and the design of antibody therapeutics with enhanced efficacy. |
BibTeX:
@article{Diebolder2014,
author = {Diebolder, Christoph A. and Beurskens, Frank J. and de Jong, Rob N. and Koning, Roman I. and Strumane, Kristin and Lindorfer, Margaret A. and Voorhorst, Marleen and Ugurlar, Deniz and Rosati, Sara and Heck, Albert J R. and van de Winkel, Jan G J. and Wilson, Ian A. and Koster, Abraham J. and Taylor, Ronald P. and Saphire, Erica Ollmann and Burton, Dennis R. and Schuurman, Janine and Gros, Piet and Parren, Paul W H I.},
title = {Complement is activated by IgG hexamers assembled at the cell surface.},
journal = {Science},
school = {Crystal and Structural Chemistry, Bijvoet Center for Biomolecular Research, Department of Chemistry, Faculty of Science, Utrecht University, 3584 CH Utrecht, Netherlands.},
year = {2014},
volume = {343},
number = {6176},
pages = {1260--1263},
url = {http://dx.doi.org/10.1126/science.1248943},
doi = {10.1126/science.1248943}
}
|
| Diebolder CA, Halff EF, Koster AJ, Huizinga EG and Koning RI (2015), "Cryoelectron Tomography of the NAIP5/NLRC4 Inflammasome: Implications for NLR Activation.", Structure. Vol. 23(12), pp. 2349-2357. |
| Abstract: Inflammasomes are high molecular weight protein complexes that play a crucial role in innate immunity by activating caspase-1. Inflammasome formation is initiated when molecules originating from invading microorganisms activate nucleotide-binding domain and leucine-rich repeat-containing receptors (NLRs) and induce NLR multimerization. Little is known about the conformational changes involved in NLR activation and the structural organization of NLR multimers. Here, we show by cryoelectron tomography that flagellin-induced NAIP5/NLRC4 multimers form right- and left-handed helical polymers with a diameter of 28 nm and a pitch of 6.5 nm. Subtomogram averaging produced an electron density map at 4 nm resolution, which was used for rigid body fitting of NLR subdomains derived from the crystal structure of dormant NLRC4. The resulting structural model of inflammasome-incorporated NLRC4 indicates that a prominent rotation of the LRR domain of NLRC4 is necessary for multimer formation, providing unprecedented insight into the conformational changes that accompany NLR activation. |
BibTeX:
@article{Diebolder2015,
author = {Diebolder, Christoph A. and Halff, Els F. and Koster, Abraham J. and Huizinga, Eric G. and Koning, Roman I.},
title = {Cryoelectron Tomography of the NAIP5/NLRC4 Inflammasome: Implications for NLR Activation.},
journal = {Structure},
school = {Department of Molecular Cell Biology, Leiden University Medical Center, P.O. Box 9600, 2300 RC Leiden, the Netherlands. Electronic address: r.i.koning@lumc.nl.},
year = {2015},
volume = {23},
number = {12},
pages = {2349--2357},
url = {http://dx.doi.org/10.1016/j.str.2015.10.001},
doi = {10.1016/j.str.2015.10.001}
}
|
| Dobro MJ, Oikonomou CM, Piper A, Cohen J, Guo K, Jensen T, Tadayon J, Donermeyer J, Park Y, Solis BA, Kjær A, Jewett AI, McDowall AW, Chen S, Chang Y-W, Shi J, Subramanian P, Iancu CV, Li Z, Briegel A, Tocheva EI, Pilhofer M and Jensen GJ (2017), "Uncharacterized bacterial structures revealed by electron cryotomography.", Journal of bacteriology. |
| Abstract: Electron cryotomography (ECT) can reveal the native structure and arrangement of macromolecular complexes inside intact cells. This technique has greatly advanced our understanding of the ultrastructure of bacterial cells. Rather than undifferentiated bags of enzymes, we now view bacteria as structurally complex assemblies of macromolecular machines. To date, our group has applied ECT to nearly 90 different bacterial species, collecting more than 15,000 cryotomograms. In addition to known structures, we have observed several, to our knowledge, uncharacterized features in these tomograms. Some are completely novel structures; others expand the features or species range of known structure types. Here we present a survey of these uncharacterized bacterial structures in the hopes of accelerating their identification and study, and furthering our understanding of the structural complexity of bacterial cells.IMPORTANCE Bacteria are more structurally complex than is commonly appreciated and we present here a number of interesting structures that will initiate new lines of research investigating their identities and roles. |
BibTeX:
@article{Dobro2017,
author = {Dobro, Megan J and Oikonomou, Catherine M and Piper, Aidan and Cohen, John and Guo, Kylie and Jensen, Taylor and Tadayon, Jahan and Donermeyer, Joseph and Park, Yeram and Solis, Benjamin A and Kjær, Andreas and Jewett, Andrew I and McDowall, Alasdair W and Chen, Songye and Chang, Yi-Wei and Shi, Jian and Subramanian, Poorna and Iancu, Cristina V and Li, Zhuo and Briegel, Ariane and Tocheva, Elitza I and Pilhofer, Martin and Jensen, Grant J},
title = {Uncharacterized bacterial structures revealed by electron cryotomography.},
journal = {Journal of bacteriology},
year = {2017},
doi = {10.1128/JB.00100-17}
}
|
| Dudkina NV, Oostergetel GT, Lewejohann D, Braun H-P and Boekema EJ (2010), "Row-like organization of ATP synthase in intact mitochondria determined by cryo-electron tomography.", Biochim Biophys Acta. Vol. 1797(2), pp. 272-277. |
| Abstract: The fine structure of intact, close-to-spherical mitochondria from the alga Polytomella was visualized by dual-axis cryo-electron tomography. The supramolecular organization of dimeric ATP synthase in the cristae membranes was investigated by averaging subvolumes of tomograms and 3D details at approximately 6 nm resolution were revealed. Oligomeric ATP synthase is composed of rows of dimers at 12 nm intervals; the dimers make a slight angle along the row. In addition, the main features of monomeric ATP synthase, such as the conically shaped F(1) headpiece, central stalk and stator were revealed. This demonstrates the capability of dual-axis electron tomography to unravel details of proteins and their interactions in complete organelles. |
BibTeX:
@article{Dudkina2010a,
author = {Dudkina, Natalya V. and Oostergetel, Gert T. and Lewejohann, Dagmar and Braun, Hans-Peter and Boekema, Egbert J.},
title = {Row-like organization of ATP synthase in intact mitochondria determined by cryo-electron tomography.},
journal = {Biochim Biophys Acta},
school = {Electron microscopy group, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Groningen, The Netherlands.},
year = {2010},
volume = {1797},
number = {2},
pages = {272--277},
url = {http://dx.doi.org/10.1016/j.bbabio.2009.11.004},
doi = {10.1016/j.bbabio.2009.11.004}
}
|
| Dymek EE, Lin J, Fu G, Porter ME, Nicastro D and Smith EF (2019), "PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility.", Molecular biology of the cell. Vol. 30, pp. 1805-1816. |
| Abstract: We previously demonstrated that PACRG plays a role in regulating dynein-driven microtubule sliding in motile cilia. To expand our understanding of the role of PACRG in ciliary assembly and motility, we used a combination of functional and structural studies, including newly identified mutants. Using cryo-electron tomography we show that PACRG and FAP20 form the inner junction between the A- and B-tubule along the length of all nine ciliary doublet microtubules. The lack of PACRG and FAP20 also results in reduced assembly of inner-arm dynein IDA and the beak-MIP structures. In addition, our functional studies reveal that loss of PACRG and/or FAP20 causes severe cell motility defects and reduced in vitro microtubule sliding velocities. Interestingly, the addition of exogenous PACRG and/or FAP20 protein to isolated mutant axonemes restores microtubule sliding velocities, but not ciliary beating. Taken together, these studies show that PACRG and FAP20 comprise the inner junction bridge that serves as a hub for both directly modulating dynein-driven microtubule sliding, as well as for the assembly of additional ciliary components that play essential roles in generating coordinated ciliary beating. |
BibTeX:
@article{Dymek2019,
author = {Dymek, Erin E and Lin, Jianfeng and Fu, Gang and Porter, Mary E and Nicastro, Daniela and Smith, Elizabeth F},
title = {PACRG and FAP20 form the inner junction of axonemal doublet microtubules and regulate ciliary motility.},
journal = {Molecular biology of the cell},
year = {2019},
volume = {30},
pages = {1805--1816},
doi = {10.1091/mbc.E19-01-0063}
}
|
| Effantin G, Estrozi LF, Aschman N, Renesto P, Stanke N, Lindemann D, Schoehn G and Weissenhorn W (2016), "Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.", PLoS pathogens. Vol. 12, pp. e1005721. |
| Abstract: Foamy viruses (FV) belong to the genus Spumavirus, which forms a distinct lineage in the Retroviridae family. Although the infection in natural hosts and zoonotic transmission to humans is asymptomatic, FVs can replicate well in human cells making it an attractive gene therapy vector candidate. Here we present cryo-electron microscopy and (cryo-)electron tomography ultrastructural data on purified prototype FV (PFV) and PFV infected cells. Mature PFV particles have a distinct morphology with a capsid of constant dimension as well as a less ordered shell of density between the capsid and the membrane likely formed by the Gag N-terminal domain and the cytoplasmic part of the Env leader peptide gp18LP. The viral membrane contains trimeric Env glycoproteins partly arranged in interlocked hexagonal assemblies. In situ 3D reconstruction by subtomogram averaging of wild type Env and of a Env gp48TM- gp80SU cleavage site mutant showed a similar spike architecture as well as stabilization of the hexagonal lattice by clear connections between lower densities of neighboring trimers. Cryo-EM was employed to obtain a 9 Å resolution map of the glycoprotein in its pre-fusion state, which revealed extensive trimer interactions by the receptor binding subunit gp80SU at the top of the spike and three central helices derived from the fusion protein subunit gp48TM. The lower part of Env, presumably composed of interlaced parts of gp48TM, gp80SU and gp18LP anchors the spike at the membrane. We propose that the gp48TM density continues into three central transmembrane helices, which interact with three outer transmembrane helices derived from gp18LP. Our ultrastructural data and 9 Å resolution glycoprotein structure provide important new insights into the molecular architecture of PFV and its distinct evolutionary relationship with other members of the Retroviridae. |
BibTeX:
@article{Effantin2016,
author = {Effantin, Grégory and Estrozi, Leandro F and Aschman, Nick and Renesto, Patricia and Stanke, Nicole and Lindemann, Dirk and Schoehn, Guy and Weissenhorn, Winfried},
title = {Cryo-electron Microscopy Structure of the Native Prototype Foamy Virus Glycoprotein and Virus Architecture.},
journal = {PLoS pathogens},
year = {2016},
volume = {12},
pages = {e1005721},
doi = {10.1371/journal.ppat.1005721}
}
|
| Eisele DM, Arias DH, Fu X, Bloemsma EA, Steiner CP, Jensen RA, Rebentrost P, Eisele H, Tokmakoff A, Lloyd S, Nelson KA, Nicastro D, Knoester J and Bawendi MG (2014), "Robust excitons inhabit soft supramolecular nanotubes.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 111, pp. E3367-E3375. |
| Abstract: Nature's highly efficient light-harvesting antennae, such as those found in green sulfur bacteria, consist of supramolecular building blocks that self-assemble into a hierarchy of close-packed structures. In an effort to mimic the fundamental processes that govern nature's efficient systems, it is important to elucidate the role of each level of hierarchy: from molecule, to supramolecular building block, to close-packed building blocks. Here, we study the impact of hierarchical structure. We present a model system that mirrors nature's complexity: cylinders self-assembled from cyanine-dye molecules. Our work reveals that even though close-packing may alter the cylinders' soft mesoscopic structure, robust delocalized excitons are retained: Internal order and strong excitation-transfer interactions--prerequisites for efficient energy transport--are both maintained. Our results suggest that the cylindrical geometry strongly favors robust excitons; it presents a rational design that is potentially key to nature's high efficiency, allowing construction of efficient light-harvesting devices even from soft, supramolecular materials. |
BibTeX:
@article{Eisele2014,
author = {Eisele, Dörthe M and Arias, Dylan H and Fu, Xiaofeng and Bloemsma, Erik A and Steiner, Colby P and Jensen, Russell A and Rebentrost, Patrick and Eisele, Holger and Tokmakoff, Andrei and Lloyd, Seth and Nelson, Keith A and Nicastro, Daniela and Knoester, Jasper and Bawendi, Moungi G},
title = {Robust excitons inhabit soft supramolecular nanotubes.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2014},
volume = {111},
pages = {E3367--E3375},
doi = {10.1073/pnas.1408342111}
}
|
| Ertel KJ, Benefield D, Castaño-Diez D, Pennington JG, Horswill M, den Boon JA, Otegui MS and Ahlquist P (2017), "Cryo-electron tomography reveals novel features of a viral RNA replication compartment.", eLife. Vol. 6 |
| Abstract: Positive-strand RNA viruses, the largest genetic class of viruses, include numerous important pathogens such as Zika virus. These viruses replicate their RNA genomes in novel, membrane-bounded mini-organelles, but the organization of viral proteins and RNAs in these compartments has been largely unknown. We used cryo-electron tomography to reveal many previously unrecognized features of Flock house nodavirus (FHV) RNA replication compartments. These spherular invaginations of outer mitochondrial membranes are packed with electron-dense RNA fibrils and their volumes are closely correlated with RNA replication template length. Each spherule's necked aperture is crowned by a striking cupped ring structure containing multifunctional FHV RNA replication protein A. Subtomogram averaging of these crowns revealed twelve-fold symmetry, concentric flanking protrusions, and a central electron density. Many crowns were associated with long cytoplasmic fibrils, likely to be exported progeny RNA. These results provide new mechanistic insights into positive-strand RNA virus replication compartment structure, assembly, function and control. |
BibTeX:
@article{Ertel2017,
author = {Ertel, Kenneth J and Benefield, Desirée and Castaño-Diez, Daniel and Pennington, Janice G and Horswill, Mark and den Boon, Johan A and Otegui, Marisa S and Ahlquist, Paul},
title = {Cryo-electron tomography reveals novel features of a viral RNA replication compartment.},
journal = {eLife},
year = {2017},
volume = {6},
doi = {10.7554/eLife.25940}
}
|
| Farci D, Haniewicz P and Piano D (), "Mesoscale organization in the cell envelope of Deinococcus radiodurans". |
BibTeX:
@misc{Farci,
author = {Domenica Farci and Patrycja Haniewicz and Dario Piano},
title = {Mesoscale organization in the cell envelope of Deinococcus radiodurans},
doi = {10.1101/2022.01.29.478271}
}
|
| Floris D and Kühlbrandt W (2021), "Molecular landscape of etioplast inner membranes in higher plants.", Nature plants. Vol. 7, pp. 514-523. |
| Abstract: Etioplasts are photosynthetically inactive plastids that accumulate when light levels are too low for chloroplast maturation. The etioplast inner membrane consists of a paracrystalline tubular lattice and peripheral, disk-shaped membranes, respectively known as the prolamellar body and prothylakoids. These distinct membrane regions are connected into one continuous compartment. To date, no structures of protein complexes in or at etioplast membranes have been reported. Here, we used electron cryo-tomography to explore the molecular membrane landscape of pea and maize etioplasts. Our tomographic reconstructions show that ATP synthase monomers are enriched in the prothylakoids, and plastid ribosomes in the tubular lattice. The entire tubular lattice is covered by regular helical arrays of a membrane-associated protein, which we identified as the 37-kDa enzyme, light-dependent protochlorophyllide oxidoreductase (LPOR). LPOR is the most abundant protein in the etioplast, where it is responsible for chlorophyll biosynthesis, photoprotection and defining the membrane geometry of the prolamellar body. Based on the 9-Å-resolution volume of the subtomogram average, we propose a structural model of membrane-associated LPOR. |
BibTeX:
@article{Floris2021,
author = {Floris, Davide and Kühlbrandt, Werner},
title = {Molecular landscape of etioplast inner membranes in higher plants.},
journal = {Nature plants},
year = {2021},
volume = {7},
pages = {514--523},
doi = {10.1038/s41477-021-00896-z}
}
|
| Frazier Z, Xu M and Alber F (2017), "TomoMiner and TomoMinerCloud: A Software Platform for Large-Scale Subtomogram Structural Analysis.", Structure (London, England : 1993). Vol. 25, pp. 951-961.e2. |
| Abstract: Cryo-electron tomography (cryo-ET) captures the 3D electron density distribution of macromolecular complexes in close to native state. With the rapid advance of cryo-ET acquisition technologies, it is possible to generate large numbers (>100,000) of subtomograms, each containing a macromolecular complex. Often, these subtomograms represent a heterogeneous sample due to variations in the structure and composition of a complex in situ form or because particles are a mixture of different complexes. In this case subtomograms must be classified. However, classification of large numbers of subtomograms is a time-intensive task and often a limiting bottleneck. This paper introduces an open source software platform, TomoMiner, for large-scale subtomogram classification, template matching, subtomogram averaging, and alignment. Its scalable and robust parallel processing allows efficient classification of tens to hundreds of thousands of subtomograms. In addition, TomoMiner provides a pre-configured TomoMinerCloud computing service permitting users without sufficient computing resources instant access to TomoMiners high-performance features. |
BibTeX:
@article{Frazier2017,
author = {Frazier, Zachary and Xu, Min and Alber, Frank},
title = {TomoMiner and TomoMinerCloud: A Software Platform for Large-Scale Subtomogram Structural Analysis.},
journal = {Structure (London, England : 1993)},
year = {2017},
volume = {25},
pages = {951--961.e2},
doi = {10.1016/j.str.2017.04.016}
}
|
| Fu G, Wang Q, Phan N, Urbanska P, Joachimiak E, Lin J, Wloga D and Nicastro D (2018), "The I1 dynein-associated tether and tether head complex is a conserved regulator of ciliary motility.", Molecular biology of the cell. Vol. 29, pp. 1048-1059. |
| Abstract: Motile cilia are essential for propelling cells and moving fluids across tissues. The activity of axonemal dynein motors must be precisely coordinated to generate ciliary motility, but their regulatory mechanisms are not well understood. The tether and tether head (T/TH) complex was hypothesized to provide mechanical feedback during ciliary beating because it links the motor domains of the regulatory I1 dynein to the ciliary doublet microtubule. Combining genetic and biochemical approaches with cryoelectron tomography, we identified FAP44 and FAP43 (plus the algae-specific, FAP43-redundant FAP244) as T/TH components. WT-mutant comparisons revealed that the heterodimeric T/TH complex is required for the positional stability of the I1 dynein motor domains, stable anchoring of CK1 kinase, and proper phosphorylation of the regulatory IC138-subunit. T/TH also interacts with inner dynein arm d and radial spoke 3, another important motility regulator. The T/TH complex is a conserved regulator of I1 dynein and plays an important role in the signaling pathway that is critical for normal ciliary motility. |
BibTeX:
@article{Fu2018,
author = {Fu, Gang and Wang, Qian and Phan, Nhan and Urbanska, Paulina and Joachimiak, Ewa and Lin, Jianfeng and Wloga, Dorota and Nicastro, Daniela},
title = {The I1 dynein-associated tether and tether head complex is a conserved regulator of ciliary motility.},
journal = {Molecular biology of the cell},
year = {2018},
volume = {29},
pages = {1048--1059},
doi = {10.1091/mbc.E18-02-0142}
}
|
| Fu G, Zhao L, Dymek E, Hou Y, Song K, Phan N, Shang Z, Smith EF, Witman GB and Nicastro D (2019), "Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus.", The Journal of cell biology. Vol. 218, pp. 4236-4251. |
| Abstract: Nearly all motile cilia contain a central apparatus (CA) composed of two connected singlet microtubules with attached projections that play crucial roles in regulating ciliary motility. Defects in CA assembly usually result in motility-impaired or paralyzed cilia, which in humans causes disease. Despite their importance, the protein composition and functions of the CA projections are largely unknown. Here, we integrated biochemical and genetic approaches with cryo-electron tomography to compare the CA of wild-type with CA mutants. We identified a large (>2 MD) complex, the C1a-e-c supercomplex, that requires the PF16 protein for assembly and contains the CA components FAP76, FAP81, FAP92, and FAP216. We localized these subunits within the supercomplex using nanogold labeling and show that loss of any one of them results in impaired ciliary motility. These data provide insight into the subunit organization and 3D structure of the CA, which is a prerequisite for understanding the molecular mechanisms by which the CA regulates ciliary beating. |
BibTeX:
@article{Fu2019,
author = {Fu, Gang and Zhao, Lei and Dymek, Erin and Hou, Yuqing and Song, Kangkang and Phan, Nhan and Shang, Zhiguo and Smith, Elizabeth F and Witman, George B and Nicastro, Daniela},
title = {Structural organization of the C1a-e-c supercomplex within the ciliary central apparatus.},
journal = {The Journal of cell biology},
year = {2019},
volume = {218},
pages = {4236--4251},
doi = {10.1083/jcb.201906006}
}
|
| Gadadhar S, Alvarez Viar G, Hansen JN, Gong A, Kostarev A, Ialy-Radio C, Leboucher S, Whitfield M, Ziyyat A, Touré A, Alvarez L, Pigino G and Janke C (2021), "Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility.", Science (New York, N.Y.). Vol. 371 |
| Abstract: Posttranslational modifications of the microtubule cytoskeleton have emerged as key regulators of cellular functions, and their perturbations have been linked to a growing number of human pathologies. Tubulin glycylation modifies microtubules specifically in cilia and flagella, but its functional and mechanistic roles remain unclear. In this study, we generated a mouse model entirely lacking tubulin glycylation. Male mice were subfertile owing to aberrant beat patterns of their sperm flagella, which impeded the straight swimming of sperm cells. Using cryo-electron tomography, we showed that lack of glycylation caused abnormal conformations of the dynein arms within sperm axonemes, providing the structural basis for the observed dysfunction. Our findings reveal the importance of microtubule glycylation for controlled flagellar beating, directional sperm swimming, and male fertility. |
BibTeX:
@article{Gadadhar2021,
author = {Gadadhar, Sudarshan and Alvarez Viar, Gonzalo and Hansen, Jan Niklas and Gong, An and Kostarev, Aleksandr and Ialy-Radio, Côme and Leboucher, Sophie and Whitfield, Marjorie and Ziyyat, Ahmed and Touré, Aminata and Alvarez, Luis and Pigino, Gaia and Janke, Carsten},
title = {Tubulin glycylation controls axonemal dynein activity, flagellar beat, and male fertility.},
journal = {Science (New York, N.Y.)},
year = {2021},
volume = {371},
doi = {10.1126/science.abd4914}
}
|
| Gambelli L, Mesman R, Versantvoort W, Diebolder CA, Engel A, Evers W, Jetten MSM, Pabst M, Daum B and van Niftrik L (2021), "The Polygonal Cell Shape and Surface Protein Layer of Anaerobic Methane-Oxidizing , javax.xml.bind.JAXBElement@46e30b8f, , javax.xml.bind.JAXBElement@1b30f3d1, Bacteria.", Frontiers in microbiology. Vol. 12, pp. 766527. |
| Abstract: bacteria perform anaerobic methane oxidation coupled to nitrite reduction via an intra-aerobic pathway, producing carbon dioxide and dinitrogen gas. These diderm bacteria possess an unusual polygonal cell shape with sharp ridges that run along the cell body. Previously, a putative surface protein layer (S-layer) was observed as the outermost cell layer of these bacteria. We hypothesized that this S-layer is the determining factor for their polygonal cell shape. Therefore, we enriched the S-layer from cells and through LC-MS/MS identified a 31 kDa candidate S-layer protein, mela_00855, which had no homology to any other known protein. Antibodies were generated against a synthesized peptide derived from the mela_00855 protein sequence and used in immunogold localization to verify its identity and location. Both on thin sections of cells and in negative-stained enriched S-layer patches, the immunogold localization identified mela_00855 as the S-layer protein. Using electron cryo-tomography and sub-tomogram averaging of S-layer patches, we observed that the S-layer has a hexagonal symmetry. Cryo-tomography of whole cells showed that the S-layer and the outer membrane, but not the peptidoglycan layer and the cytoplasmic membrane, exhibited the polygonal shape. Moreover, the S-layer consisted of multiple rigid sheets that partially overlapped, most likely giving rise to the unique polygonal cell shape. These characteristics make the S-layer of a distinctive and intriguing case to study. |
BibTeX:
@article{Gambelli2021,
author = {Gambelli, Lavinia and Mesman, Rob and Versantvoort, Wouter and Diebolder, Christoph A. and Engel, Andreas and Evers, Wiel and Jetten, Mike S. M. and Pabst, Martin and Daum, Bertram and van Niftrik, Laura},
title = {The Polygonal Cell Shape and Surface Protein Layer of Anaerobic Methane-Oxidizing , javax.xml.bind.JAXBElement@46e30b8f, , javax.xml.bind.JAXBElement@1b30f3d1, Bacteria.},
journal = {Frontiers in microbiology},
year = {2021},
volume = {12},
pages = {766527},
doi = {10.3389/fmicb.2021.766527}
}
|
| Gambelli L, Meyer BH, McLaren M, Sanders K, Quax TEF, Gold VAM, Albers S-V and Daum B (2019), "Architecture and modular assembly of , javax.xml.bind.JAXBElement@71e7283b, S-layers revealed by electron cryotomography.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 116, pp. 25278-25286. |
| Abstract: Surface protein layers (S-layers) often form the only structural component of the archaeal cell wall and are therefore important for cell survival. S-layers have a plethora of cellular functions including maintenance of cell shape, osmotic, and mechanical stability, the formation of a semipermeable protective barrier around the cell, and cell-cell interaction, as well as surface adhesion. Despite the central importance of S-layers for archaeal life, their 3-dimensional (3D) architecture is still poorly understood. Here we present detailed 3D electron cryomicroscopy maps of archaeal S-layers from 3 different strains. We were able to pinpoint the positions and determine the structure of the 2 subunits SlaA and SlaB. We also present a model describing the assembly of the mature S-layer. |
BibTeX:
@article{Gambelli2019,
author = {Gambelli, Lavinia and Meyer, Benjamin H and McLaren, Mathew and Sanders, Kelly and Quax, Tessa E F and Gold, Vicki A M and Albers, Sonja-Verena and Daum, Bertram},
title = {Architecture and modular assembly of , javax.xml.bind.JAXBElement@71e7283b, S-layers revealed by electron cryotomography.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2019},
volume = {116},
pages = {25278--25286},
doi = {10.1073/pnas.1911262116}
}
|
| Ghosal D, Chang Y-W, Jeong KC, Vogel JP and Jensen GJ (2017), "In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography.", EMBO reports. Vol. 18, pp. 726-732. |
| Abstract: Type IV secretion systems (T4SSs) are large macromolecular machines that translocate protein and DNA and are involved in the pathogenesis of multiple human diseases. Here, using electron cryotomography (ECT), we report the in situ structure of the Dot/Icm type IVB secretion system (T4BSS) utilized by the human pathogen Legionella pneumophila This is the first structure of a type IVB secretion system, and also the first structure of any T4SS in situ While the Dot/Icm system shares almost no sequence similarity with type IVA secretion systems (T4ASSs), its overall structure is seen here to be remarkably similar to previously reported T4ASS structures (those encoded by the R388 plasmid in Escherichia coli and the cag pathogenicity island in Helicobacter pylori). This structural similarity suggests shared aspects of mechanism. However, compared to the negative-stain reconstruction of the purified T4ASS from the R388 plasmid, the L. pneumophila Dot/Icm system is approximately twice as long and wide and exhibits several additional large densities, reflecting type-specific elaborations and potentially better structural preservation in situ. |
BibTeX:
@article{Ghosal2017,
author = {Ghosal, Debnath and Chang, Yi-Wei and Jeong, Kwangcheol C and Vogel, Joseph P and Jensen, Grant J},
title = {In situ structure of the Legionella Dot/Icm type IV secretion system by electron cryotomography.},
journal = {EMBO reports},
year = {2017},
volume = {18},
pages = {726--732},
doi = {10.15252/embr.201643598}
}
|
| Ghosal D, Jeong KC, Chang Y-W, Gyore J, Teng L, Gardner A, Vogel JP and Jensen GJ (2019), "Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS.", Nature microbiology. Vol. 4, pp. 1173-1182. |
| Abstract: Legionella pneumophila survives and replicates inside host cells by secreting 300 effectors through the defective in organelle trafficking (Dot)/intracellular multiplication (Icm) type IVB secretion system (T4BSS). Here, we used complementary electron cryotomography and immunofluorescence microscopy to investigate the molecular architecture and biogenesis of the Dot/Icm secretion apparatus. Electron cryotomography mapped the location of the core and accessory components of the Legionella core transmembrane subcomplex, revealing a well-ordered central channel that opens into a large, windowed secretion chamber with an unusual 13-fold symmetry. Immunofluorescence microscopy deciphered an early-stage assembly process that begins with the targeting of Dot/Icm components to the bacterial poles. Polar targeting of this T4BSS is mediated by two Dot/Icm proteins, DotU and IcmF, that, interestingly, are homologues of the T6SS membrane complex components TssL and TssM, suggesting that the Dot/Icm T4BSS is a hybrid system. Together, these results revealed that the Dot/Icm complex assembles in an 'axial-to-peripheral' pattern. |
BibTeX:
@article{Ghosal2019a,
author = {Ghosal, Debnath and Jeong, Kwangcheol C and Chang, Yi-Wei and Gyore, Jacob and Teng, Lin and Gardner, Adam and Vogel, Joseph P and Jensen, Grant J},
title = {Molecular architecture, polar targeting and biogenesis of the Legionella Dot/Icm T4SS.},
journal = {Nature microbiology},
year = {2019},
volume = {4},
pages = {1173--1182},
doi = {10.1038/s41564-019-0427-4}
}
|
| Ghosal D, Kaplan M, Chang Y-W and Jensen GJ (2019), "In Situ Imaging and Structure Determination of Bacterial Toxin Delivery Systems Using Electron Cryotomography.", Methods in molecular biology (Clifton, N.J.). Vol. 1921, pp. 249-265. |
| Abstract: Determining the three-dimensional structure of biomacromolecules at high resolution in their native cellular environment is a major challenge for structural biology. Toward this end, electron cryotomography (ECT) allows large bio-macromolecular assemblies to be imaged directly in their hydrated physiological milieu to 4 nm resolution. Combining ECT with other techniques like fluorescent imaging, immunogold labeling, and genetic manipulation has allowed the in situ investigation of complex biological processes at macromolecular resolution. Furthermore, the advent of cryogenic focused ion beam (FIB) milling has extended the domain of ECT to include regions even deep within thick eukaryotic cells. Anticipating two audiences (scientists who just want to understand the potential and general workflow involved and scientists who are learning how to do the work themselves), here we present both a broad overview of this kind of work and a step-by-step example protocol for ECT and subtomogram averaging using the Legionella pneumophila Dot/Icm type IV secretion system (T4SS) as a case study. While the general workflow is presented in step-by-step detail, we refer to online tutorials, user's manuals, and other training materials for the essential background understanding needed to perform each step. |
BibTeX:
@article{Ghosal2019,
author = {Ghosal, Debnath and Kaplan, Mohammed and Chang, Yi-Wei and Jensen, Grant J},
title = {In Situ Imaging and Structure Determination of Bacterial Toxin Delivery Systems Using Electron Cryotomography.},
journal = {Methods in molecular biology (Clifton, N.J.)},
year = {2019},
volume = {1921},
pages = {249--265},
doi = {10.1007/978-1-4939-9048-1_16}
}
|
| Ghosal D, Kim KW, Zheng H, Kaplan M, Truchan HK, Lopez AE, McIntire IE, Vogel JP, Cianciotto NP and Jensen GJ (2019), "In vivo structure of the Legionella type II secretion system by electron cryotomography.", Nature microbiology. Vol. 4, pp. 2101-2108. |
| Abstract: The type II secretion system (T2SS) is a multiprotein envelope-spanning assembly that translocates a wide range of virulence factors, enzymes and effectors through the outer membrane of many Gram-negative bacteria . Here, using electron cryotomography and subtomogram averaging methods, we reveal the in vivo structure of an intact T2SS imaged within the human pathogen Legionella pneumophila. Although the T2SS has only limited sequence and component homology with the evolutionarily related type IV pilus (T4P) system , we show that their overall architectures are remarkably similar. Despite similarities, there are also differences, including, for example, that the T2SS-ATPase complex is usually present but disengaged from the inner membrane, the T2SS has a much longer periplasmic vestibule and it has a short-lived flexible pseudopilus. Placing atomic models of the components into our electron cryotomography map produced a complete architectural model of the intact T2SS that provides insights into the structure and function of its components, its position within the cell envelope and the interactions between its different subcomplexes. |
BibTeX:
@article{Ghosal2019b,
author = {Ghosal, Debnath and Kim, Ki Woo and Zheng, Huaixin and Kaplan, Mohammed and Truchan, Hilary K and Lopez, Alberto E and McIntire, Ian E and Vogel, Joseph P and Cianciotto, Nicholas P and Jensen, Grant J},
title = {In vivo structure of the Legionella type II secretion system by electron cryotomography.},
journal = {Nature microbiology},
year = {2019},
volume = {4},
pages = {2101--2108},
doi = {10.1038/s41564-019-0603-6}
}
|
| Gibbons BJ, Brignole EJ, Azubel M, Murakami K, Voss NR, Bushnell DA, Asturias FJ and Kornberg RD (2012), "Subunit architecture of general transcription factor TFIIH", Proceedings of the National Academy of Sciences. Vol. 109(6), pp. 1949-1954. National Acad Sciences.
[BibTeX] |
BibTeX:
@article{Gibbons2012,
author = {Gibbons, Brian J and Brignole, Edward J and Azubel, Maia and Murakami, Kenji and Voss, Neil R and Bushnell, David A and Asturias, Francisco J and Kornberg, Roger D},
title = {Subunit architecture of general transcription factor TFIIH},
journal = {Proceedings of the National Academy of Sciences},
publisher = {National Acad Sciences},
year = {2012},
volume = {109},
number = {6},
pages = {1949--1954}
}
|
| Gipson P, Fukuda Y, Danev R, Lai Y, Chen D-H, Baumeister W and Brunger AT (2017), "Morphologies of synaptic protein membrane fusion interfaces.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 114, pp. 9110-9115. |
| Abstract: Neurotransmitter release is orchestrated by synaptic proteins, such as SNAREs, synaptotagmin, and complexin, but the molecular mechanisms remain unclear. We visualized functionally active synaptic proteins reconstituted into proteoliposomes and their interactions in a native membrane environment by electron cryotomography with a Volta phase plate for improved resolvability. The images revealed individual synaptic proteins and synaptic protein complex densities at prefusion contact sites between membranes. We observed distinct morphologies of individual synaptic proteins and their complexes. The minimal system, consisting of neuronal SNAREs and synaptotagmin-1, produced point and long-contact prefusion states. Morphologies and populations of these states changed as the regulatory factors complexin and Munc13 were added. Complexin increased the membrane separation, along with a higher propensity of point contacts. Further inclusion of the priming factor Munc13 exclusively restricted prefusion states to point contacts, all of which efficiently fused upon Ca2+ triggering. We conclude that synaptic proteins have evolved to limit possible contact site assemblies and morphologies to those that promote fast Ca2+-triggered release. |
BibTeX:
@article{Gipson2017,
author = {Gipson, Preeti and Fukuda, Yoshiyuki and Danev, Radostin and Lai, Ying and Chen, Dong-Hua and Baumeister, Wolfgang and Brunger, Axel T},
title = {Morphologies of synaptic protein membrane fusion interfaces.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2017},
volume = {114},
pages = {9110--9115},
doi = {10.1073/pnas.1708492114}
}
|
| Gold VA, Chroscicki P, Bragoszewski P and Chacinska A (2017), "Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography.", EMBO reports. Vol. 18, pp. 1786-1800. |
| Abstract: We employed electron cryo-tomography to visualize cytosolic ribosomes on the surface of mitochondria. Translation-arrested ribosomes reveal the clustered organization of the TOM complex, corroborating earlier reports of localized translation. Ribosomes are shown to interact specifically with the TOM complex, and nascent chain binding is crucial for ribosome recruitment and stabilization. Ribosomes are bound to the membrane in discrete clusters, often in the vicinity of the crista junctions. This interaction highlights how protein synthesis may be coupled with transport. Our work provides unique insights into the spatial organization of cytosolic ribosomes on mitochondria. |
BibTeX:
@article{Gold2017,
author = {Gold, Vicki Am and Chroscicki, Piotr and Bragoszewski, Piotr and Chacinska, Agnieszka},
title = {Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography.},
journal = {EMBO reports},
year = {2017},
volume = {18},
pages = {1786--1800},
doi = {10.15252/embr.201744261}
}
|
| Gold VAM, Ieva R, Walter A, Pfanner N, van der Laan M and Kühlbrandt W (2014), "Visualizing active membrane protein complexes by electron cryotomography.", Nature communications. Vol. 5, pp. 4129. |
| Abstract: Unravelling the structural organization of membrane protein machines in their active state and native lipid environment is a major challenge in modern cell biology research. Here we develop the STAMP (Specifically TArgeted Membrane nanoParticle) technique as a strategy to localize protein complexes in situ by electron cryotomography (cryo-ET). STAMP selects active membrane protein complexes and marks them with quantum dots. Taking advantage of new electron detector technology that is currently revolutionizing cryotomography in terms of achievable resolution, this approach enables us to visualize the three-dimensional distribution and organization of protein import sites in mitochondria. We show that import sites cluster together in the vicinity of crista membranes, and we reveal unique details of the mitochondrial protein import machinery in action. STAMP can be used as a tool for site-specific labelling of a multitude of membrane proteins by cryo-ET in the future. |
BibTeX:
@article{Gold2014,
author = {Gold, Vicki A M and Ieva, Raffaele and Walter, Andreas and Pfanner, Nikolaus and van der Laan, Martin and Kühlbrandt, Werner},
title = {Visualizing active membrane protein complexes by electron cryotomography.},
journal = {Nature communications},
year = {2014},
volume = {5},
pages = {4129},
doi = {10.1038/ncomms5129}
}
|
| Gold VAM, Salzer R, Averhoff B and Kühlbrandt W (2015), "Structure of a type IV pilus machinery in the open and closed state.", eLife. Vol. 4 |
| Abstract: Proteins of the secretin family form large macromolecular complexes, which assemble in the outer membrane of Gram-negative bacteria. Secretins are major components of type II and III secretion systems and are linked to extrusion of type IV pili (T4P) and to DNA uptake. By electron cryo-tomography of whole Thermus thermophilus cells, we determined the in situ structure of a T4P molecular machine in the open and the closed state. Comparison reveals a major conformational change whereby the N-terminal domains of the central secretin PilQ shift by 30 Å, and two periplasmic gates open to make way for pilus extrusion. Furthermore, we determine the structure of the assembled pilus. |
BibTeX:
@article{Gold2015,
author = {Gold, Vicki A M and Salzer, Ralf and Averhoff, Beate and Kühlbrandt, Werner},
title = {Structure of a type IV pilus machinery in the open and closed state.},
journal = {eLife},
year = {2015},
volume = {4},
doi = {10.7554/eLife.07380}
}
|
| Grange M, Vasishtan D and Grünewald K (2017), "Cellular electron cryo tomography and in situ sub-volume averaging reveal the context of microtubule-based processes.", Journal of structural biology. Vol. 197, pp. 181-190. |
| Abstract: Electron cryo-tomography (cryoET) is currently the only technique that allows the direct observation of proteins in their native cellular environment. Sub-volume averaging of electron tomograms offers a route to increase the signal-to-noise of repetitive biological structures, such improving the information content and interpretability of tomograms. We discuss the potential for sub-volume averaging in highlighting and investigating specific processes in situ, focusing on microtubule structure and viral infection. We show that (i) in situ sub-volume averaging from single tomograms can guide and complement segmentation of biological features, (ii) the in situ determination of the structure of individual viruses is possible as they infect a cell, and (iii) novel, transient processes can be imaged with high levels of detail. |
BibTeX:
@article{Grange2017,
author = {Grange, Michael and Vasishtan, Daven and Grünewald, Kay},
title = {Cellular electron cryo tomography and in situ sub-volume averaging reveal the context of microtubule-based processes.},
journal = {Journal of structural biology},
year = {2017},
volume = {197},
pages = {181--190},
doi = {10.1016/j.jsb.2016.06.024}
}
|
| Gui L, Song K, Tritschler D, Bower R, Yan S, Dai A, Augspurger K, Sakizadeh J, Grzemska M, Ni T, Porter ME and Nicastro D (2019), "Scaffold subunits support associated subunit assembly in the , javax.xml.bind.JAXBElement@123d407, ciliary nexin-dynein regulatory complex.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 116, pp. 23152-23162. |
| Abstract: The nexin-dynein regulatory complex (N-DRC) in motile cilia and flagella functions as a linker between neighboring doublet microtubules, acts to stabilize the axonemal core structure, and serves as a central hub for the regulation of ciliary motility. Although the N-DRC has been studied extensively using genetic, biochemical, and structural approaches, the precise arrangement of the 11 (or more) N-DRC subunits remains unknown. Here, using cryo-electron tomography, we have compared the structure of wild-type flagella to that of strains with specific DRC subunit deletions or rescued strains with tagged DRC subunits. Our results show that DRC7 is a central linker subunit that helps connect the N-DRC to the outer dynein arms. DRC11 is required for the assembly of DRC8, and DRC8/11 form a subcomplex in the proximal lobe of the linker domain that is required to form stable contacts to the neighboring B-tubule. Gold labeling of tagged subunits determines the precise locations of the previously ambiguous N terminus of DRC4 and C terminus of DRC5. DRC4 is now shown to contribute to the core scaffold of the N-DRC. Our results reveal the overall architecture of N-DRC, with the 3 subunits DRC1/2/4 forming a core complex that serves as the scaffold for the assembly of the "functional subunits," namely DRC3/5-8/11. These findings shed light on N-DRC assembly and its role in regulating flagellar beating. |
BibTeX:
@article{Gui2019,
author = {Gui, Long and Song, Kangkang and Tritschler, Douglas and Bower, Raqual and Yan, Si and Dai, Aguang and Augspurger, Katherine and Sakizadeh, Jason and Grzemska, Magdalena and Ni, Thomas and Porter, Mary E and Nicastro, Daniela},
title = {Scaffold subunits support associated subunit assembly in the , javax.xml.bind.JAXBElement@123d407, ciliary nexin-dynein regulatory complex.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2019},
volume = {116},
pages = {23152--23162},
doi = {10.1073/pnas.1910960116}
}
|
| Gui M, Croft JT, Zabeo D, Acharya V, Kollman JM, Burgoyne T, Höög JL and Brown A (2022), "SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules" Vol. 119 |
BibTeX:
@article{Gui2022,
author = {Miao Gui and Jacob T. Croft and Davide Zabeo and Vajradhar Acharya and Justin M. Kollman and Thomas Burgoyne and Johanna L. Höög and Alan Brown},
title = {SPACA9 is a lumenal protein of human ciliary singlet and doublet microtubules},
year = {2022},
volume = {119},
doi = {10.1073/pnas.2207605119}
}
|
| Guichard P, Hamel V, Neves A and Gönczy P (2015), "Isolation, cryotomography, and three-dimensional reconstruction of centrioles.", Methods Cell Biol. Vol. 129, pp. 191-209. |
| Abstract: Centrioles and basal bodies (referred to hereafter as centrioles for simplicity) are microtubule-based cylindrical organelles that are typically ∼450-nm long and ∼250nm in diameter. The centriole is composed of three distinct regions: the distal part characterized by microtubule doublets, the central core that harbors microtubule triplets, which are also present in the proximal part that also contains the cartwheel, a structure crucial for centriole assembly. The cartwheel was initially revealed by conventional electron microscopy of resin-embedded samples and is thought to impart the near universal ninefold symmetry of centrioles. Deciphering the native architecture of the cartwheel has proven challenging owing to its small dimensions and the difficulties in isolating it. Here, we present a method to purify and analyze the structure of the exceptionally long Trichonympha centriole by cryotomography and subtomogram averaging. Using this method, we revealed the native architecture of the proximal cartwheel-containing region at ∼40Å-resolution. This method can be applied as a general strategy for uncovering the structure of centrioles in other species. |
BibTeX:
@article{Guichard2015,
author = {Guichard, Paul and Hamel, Virginie and Neves, Aitana and Gönczy, Pierre},
title = {Isolation, cryotomography, and three-dimensional reconstruction of centrioles.},
journal = {Methods Cell Biol},
school = {Swiss Institute for Experimental Cancer Research (ISREC), School of Life Sciences, Swiss Federal Institute of Technology (EPFL), Lausanne, Switzerland.},
year = {2015},
volume = {129},
pages = {191--209},
url = {http://dx.doi.org/10.1016/bs.mcb.2015.04.003},
doi = {10.1016/bs.mcb.2015.04.003}
}
|
| Guyomar C, Bousquet C, Ku S, Heumann JM, Guilloux G, Gaillard N, Heichette C, Duchesne L, Steinmetz MO, Gibeaux R and Chrétien D (), "Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts" Vol. 11 |
BibTeX:
@article{Guyomar,
author = {Charlotte Guyomar and Clément Bousquet and Siou Ku and John M. Heumann and Gabriel Guilloux and Natacha Gaillard and Claire Heichette and Laurence Duchesne and Michel O. Steinmetz and Romain Gibeaux and Denis Chrétien},
title = {Changes in seam number and location induce holes within microtubules assembled from porcine brain tubulin and in Xenopus egg cytoplasmic extracts},
volume = {11},
doi = {10.7554/elife.83021}
}
|
| Hagen C, Dent KC, Zeev-Ben-Mordehai T, Grange M, Bosse JB, Whittle C, Klupp BG, Siebert CA, Vasishtan D, Bäuerlein FJB, Cheleski J, Werner S, Guttmann P, Rehbein S, Henzler K, Demmerle J, Adler B, Koszinowski U, Schermelleh L, Schneider G, Enquist LW, Plitzko JM, Mettenleiter TC and Grünewald K (2015), "Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.", Cell. Vol. 163(7), pp. 1692-1701. |
| Abstract: Vesicular nucleo-cytoplasmic transport is becoming recognized as a general cellular mechanism for translocation of large cargoes across the nuclear envelope. Cargo is recruited, enveloped at the inner nuclear membrane (INM), and delivered by membrane fusion at the outer nuclear membrane. To understand the structural underpinning for this trafficking, we investigated nuclear egress of progeny herpesvirus capsids where capsid envelopment is mediated by two viral proteins, forming the nuclear egress complex (NEC). Using a multi-modal imaging approach, we visualized the NEC in situ forming coated vesicles of defined size. Cellular electron cryo-tomography revealed a protein layer showing two distinct hexagonal lattices at its membrane-proximal and membrane-distant faces, respectively. NEC coat architecture was determined by combining this information with integrative modeling using small-angle X-ray scattering data. The molecular arrangement of the NEC establishes the basic mechanism for budding and scission of tailored vesicles at the INM. |
BibTeX:
@article{Hagen2015,
author = {Hagen, Christoph and Dent, Kyle C. and Zeev-Ben-Mordehai, Tzviya and Grange, Michael and Bosse, Jens B. and Whittle, Cathy and Klupp, Barbara G. and Siebert, C Alistair and Vasishtan, Daven and Bäuerlein, Felix J B. and Cheleski, Juliana and Werner, Stephan and Guttmann, Peter and Rehbein, Stefan and Henzler, Katja and Demmerle, Justin and Adler, Barbara and Koszinowski, Ulrich and Schermelleh, Lothar and Schneider, Gerd and Enquist, Lynn W. and Plitzko, Jürgen M. and Mettenleiter, Thomas C. and Grünewald, Kay},
title = {Structural Basis of Vesicle Formation at the Inner Nuclear Membrane.},
journal = {Cell},
school = {Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Roosevelt Drive, Oxford OX3 7BN, UK. Electronic address: kay@strubi.ox.ac.uk.},
year = {2015},
volume = {163},
number = {7},
pages = {1692--1701},
url = {http://dx.doi.org/10.1016/j.cell.2015.11.029},
doi = {10.1016/j.cell.2015.11.029}
}
|
| Haglin ER, Yang W, Briegel A and Thompson LK (2017), "His-Tag-Mediated Dimerization of Chemoreceptors Leads to Assembly of Functional Nanoarrays.", Biochemistry. Vol. 56, pp. 5874-5885. |
| Abstract: Transmembrane chemotaxis receptors are found in bacteria in extended hexagonal arrays stabilized by the membrane and by cytosolic binding partners, the kinase CheA and coupling protein CheW. Models of array architecture and assembly propose receptors cluster into trimers of dimers that associate with one CheA dimer and two CheW monomers to form the minimal "core unit" necessary for signal transduction. Reconstructing in vitro chemoreceptor ternary complexes that are homogeneous and functional and exhibit native architecture remains a challenge. Here we report that His-tag-mediated receptor dimerization with divalent metals is sufficient to drive assembly of nativelike functional arrays of a receptor cytoplasmic fragment. Our results indicate receptor dimerization initiates assembly and precedes formation of ternary complexes with partial kinase activity. Restoration of maximal kinase activity coincides with a shift to larger complexes, suggesting that kinase activity depends on interactions beyond the core unit. We hypothesize that achieving maximal activity requires building core units into hexagons and/or coalescing hexagons into the extended lattice. Overall, the minimally perturbing His-tag-mediated dimerization leads to assembly of chemoreceptor arrays with native architecture and thus serves as a powerful tool for studying the assembly and mechanism of this complex and other multiprotein complexes. |
BibTeX:
@article{Haglin2017,
author = {Haglin, Elizabeth R and Yang, Wen and Briegel, Ariane and Thompson, Lynmarie K},
title = {His-Tag-Mediated Dimerization of Chemoreceptors Leads to Assembly of Functional Nanoarrays.},
journal = {Biochemistry},
year = {2017},
volume = {56},
pages = {5874--5885},
doi = {10.1021/acs.biochem.7b00698}
}
|
| Hampton CM, Strauss JD, Ke Z, Dillard RS, Hammonds JE, Alonas E, Desai TM, Marin M, Storms RE, Leon F, Melikyan GB, Santangelo PJ, Spearman PW and Wright ER (2017), "Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.", Nature protocols. Vol. 12, pp. 150-167. |
| Abstract: Correlative light and electron microscopy (CLEM) combines spatiotemporal information from fluorescence light microscopy (fLM) with high-resolution structural data from cryo-electron tomography (cryo-ET). These technologies provide opportunities to bridge knowledge gaps between cell and structural biology. Here we describe our protocol for correlated cryo-fLM, cryo-electron microscopy (cryo-EM), and cryo-ET (i.e., cryo-CLEM) of virus-infected or transfected mammalian cells. Mammalian-derived cells are cultured on EM substrates, using optimized conditions that ensure that the cells are spread thinly across the substrate and are not physically disrupted. The cells are then screened by fLM and vitrified before acquisition of cryo-fLM and cryo-ET images, which is followed by data processing. A complete session from grid preparation through data collection and processing takes 5-15 d for an individual experienced in cryo-EM. |
BibTeX:
@article{Hampton2017,
author = {Hampton, Cheri M and Strauss, Joshua D and Ke, Zunlong and Dillard, Rebecca S and Hammonds, Jason E and Alonas, Eric and Desai, Tanay M and Marin, Mariana and Storms, Rachel E and Leon, Fredrick and Melikyan, Gregory B and Santangelo, Philip J and Spearman, Paul W and Wright, Elizabeth R},
title = {Correlated fluorescence microscopy and cryo-electron tomography of virus-infected or transfected mammalian cells.},
journal = {Nature protocols},
year = {2017},
volume = {12},
pages = {150--167},
doi = {10.1038/nprot.2016.168}
}
|
| Han L, Rao Q, Yang R, Wang Y, Chai P, Xiong Y and Zhang K (), "Cryo-EM structure of an active central apparatus". |
BibTeX:
@misc{Han,
author = {Long Han and Qinhui Rao and Renbin Yang and Yue Wang and Pengxin Chai and Yong Xiong and Kai Zhang},
title = {Cryo-EM structure of an active central apparatus},
doi = {10.1101/2022.01.23.477438}
}
|
| Hartman R, Eilers BJ, Bollschweiler D, Munson-McGee JH, Engelhardt H, Young MJ and Lawrence CM (2019), "The Molecular Mechanism of Cellular Attachment for an Archaeal Virus.", Structure (London, England : 1993). Vol. 27, pp. 1634-1646.e3. |
| Abstract: Sulfolobus turreted icosahedral virus (STIV) is a model archaeal virus and member of the PRD1-adenovirus lineage. Although STIV employs pyramidal lysis structures to exit the host, knowledge of the viral entry process is lacking. We therefore initiated studies on STIV attachment and entry. Negative stain and cryoelectron micrographs showed virion attachment to pili-like structures emanating from the Sulfolobus host. Tomographic reconstruction and sub-tomogram averaging revealed pili recognition by the STIV C381 turret protein. Specifically, the triple jelly roll structure of C381 determined by X-ray crystallography shows that pilus recognition is mediated by conserved surface residues in the second and third domains. In addition, the STIV petal protein (C557), when present, occludes the pili binding site, suggesting that it functions as a maturation protein. Combined, these results demonstrate a role for the namesake STIV turrets in initial cellular attachment and provide the first molecular model for viral attachment in the archaeal domain of life. |
BibTeX:
@article{Hartman2019,
author = {Hartman, Ross and Eilers, Brian J and Bollschweiler, Daniel and Munson-McGee, Jacob H and Engelhardt, Harald and Young, Mark J and Lawrence, C Martin},
title = {The Molecular Mechanism of Cellular Attachment for an Archaeal Virus.},
journal = {Structure (London, England : 1993)},
year = {2019},
volume = {27},
pages = {1634--1646.e3},
doi = {10.1016/j.str.2019.09.005}
}
|
| Henderson LD and Beeby M (2018), "High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly.", Methods in molecular biology (Clifton, N.J.). Vol. 1764, pp. 29-44. |
| Abstract: Electron cryo-tomography and subtomogram averaging enable visualization of protein complexes in situ, in three dimensions, in a near-native frozen-hydrated state to nanometer resolutions. To achieve this, intact cells are vitrified and imaged over a range of tilts within an electron microscope. These images can subsequently be reconstructed into a three-dimensional volume representation of the sample cell. Because complexes are visualized in situ, crucial insights into their mechanism, assembly process, and dynamic interactions with other proteins become possible. To illustrate the electron cryo-tomography workflow for visualizing protein complexes in situ, we describe our workflow of preparing samples, imaging, and image processing using Leginon for data collection, IMOD for image reconstruction, and PEET for subtomogram averaging. |
BibTeX:
@article{Henderson2018,
author = {Henderson, Louie D and Beeby, Morgan},
title = {High-Throughput Electron Cryo-tomography of Protein Complexes and Their Assembly.},
journal = {Methods in molecular biology (Clifton, N.J.)},
year = {2018},
volume = {1764},
pages = {29--44},
doi = {10.1007/978-1-4939-7759-8_2}
}
|
| Hetzel U, Sironen T, Laurinmäki P, Liljeroos L, Patjas A, Henttonen H, Vaheri A, Artelt A, Kipar A, Butcher SJ, Vapalahti O and Hepojoki J (2013), "Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease.", J Virol. Vol. 87(20), pp. 10918-10935. |
| Abstract: Boid inclusion body disease (BIBD) is a progressive, usually fatal disease of constrictor snakes, characterized by cytoplasmic inclusion bodies (IB) in a wide range of cell types. To identify the causative agent of the disease, we established cell cultures from BIBD-positive and -negative boa constrictors. The IB phenotype was maintained in cultured cells of affected animals, and supernatants from these cultures caused the phenotype in cultures originating from BIBD-negative snakes. Viruses were purified from the supernatants by ultracentrifugation and subsequently identified as arenaviruses. Purified virus also induced the IB phenotype in naive cells, which fulfilled Koch's postulates in vitro. One isolate, tentatively designated University of Helsinki virus (UHV), was studied in depth. Sequencing confirmed that UHV is a novel arenavirus species that is distinct from other known arenaviruses including those recently identified in snakes with BIBD. The morphology of UHV was established by cryoelectron tomography and subtomographic averaging, revealing the trimeric arenavirus spike structure at 3.2-nm resolution. Immunofluorescence, immunohistochemistry, and immunoblotting with a polyclonal rabbit antiserum against UHV and reverse transcription-PCR (RT-PCR) revealed the presence of genetically diverse arenaviruses in a large cohort of snakes with BIBD, confirming the causative role of arenaviruses. Some snakes were also found to carry arenavirus antibodies. Furthermore, mammalian cells (Vero E6) were productively infected with UHV, demonstrating the potential of arenaviruses to cross species barriers. In conclusion, we propose the newly identified lineage of arenaviruses associated with BIBD as a novel taxonomic entity, boid inclusion body disease-associated arenaviruses (BIBDAV), in the family Arenaviridae. |
BibTeX:
@article{Hetzel2013,
author = {Hetzel, Udo and Sironen, Tarja and Laurinmäki, Pasi and Liljeroos, Lassi and Patjas, Aino and Henttonen, Heikki and Vaheri, Antti and Artelt, Annette and Kipar, Anja and Butcher, Sarah J. and Vapalahti, Olli and Hepojoki, Jussi},
title = {Isolation, identification, and characterization of novel arenaviruses, the etiological agents of boid inclusion body disease.},
journal = {J Virol},
school = {Department of Veterinary Biosciences, Faculty of Veterinary Medicine, University of Helsinki, Helsinki, Finland.},
year = {2013},
volume = {87},
number = {20},
pages = {10918--10935},
url = {http://dx.doi.org/10.1128/JVI.01123-13},
doi = {10.1128/JVI.01123-13}
}
|
| Heumann JM, Hoenger A and Mastronarde DN (2011), "Clustering and variance maps for cryo-electron tomography using wedge-masked differences.", J Struct Biol. Vol. 175(3), pp. 288-299. |
| Abstract: Cryo-electron tomography provides 3D imaging of frozen hydrated biological samples with nanometer resolution. Reconstructed volumes suffer from low signal-to-noise-ratio (SNR)(1) and artifacts caused by systematically missing tomographic data. Both problems can be overcome by combining multiple subvolumes with varying orientations, assuming they contain identical structures. Clustering (unsupervised classification) is required to ensure or verify population homogeneity, but this process is complicated by the problems of poor SNR and missing data, the factors that led to consideration of multiple subvolumes in the first place. Here, we describe a new approach to clustering and variance mapping in the face of these difficulties. The combined subvolume is taken as an estimate of the true subvolume, and the effect of missing data is computed for individual subvolumes. Clustering and variance mapping then proceed based on differences between expected and observed subvolumes. We show that this new method is faster and more accurate than two current, widely used techniques. |
BibTeX:
@article{Heumann2011,
author = {Heumann, John M. and Hoenger, Andreas and Mastronarde, David N.},
title = {Clustering and variance maps for cryo-electron tomography using wedge-masked differences.},
journal = {J Struct Biol},
school = {Boulder Laboratory For 3D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA. john.heumann@colorado.edu},
year = {2011},
volume = {175},
number = {3},
pages = {288--299},
url = {http://dx.doi.org/10.1016/j.jsb.2011.05.011},
doi = {10.1016/j.jsb.2011.05.011}
}
|
| Heuser T, Barber CF, Lin J, Krell J, Rebesco M, Porter ME and Nicastro D (2012), "Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein.", Proc Natl Acad Sci U S A. Vol. 109(30), pp. E2067-E2076. |
| Abstract: Cilia and flagella are highly conserved motile and sensory organelles in eukaryotes, and defects in ciliary assembly and motility cause many ciliopathies. The two-headed I1 inner arm dynein is a critical regulator of ciliary and flagellar beating. To understand I1 architecture and function better, we analyzed the 3D structure and composition of the I1 dynein in Chlamydomonas axonemes by cryoelectron tomography and subtomogram averaging. Our data revealed several connections from the I1 dynein to neighboring structures that are likely to be important for assembly and/or regulation, including a tether linking one I1 motor domain to the doublet microtubule and doublet-specific differences potentially contributing to the asymmetrical distribution of dynein activity required for ciliary beating. We also imaged three I1 mutants and analyzed their polypeptide composition using 2D gel-based proteomics. Structural and biochemical comparisons revealed the likely location of the regulatory IC138 phosphoprotein and its associated subcomplex. Overall, our studies demonstrate that I1 dynein is connected to multiple structures within the axoneme, and therefore ideally positioned to integrate signals that regulate ciliary motility. |
BibTeX:
@article{Heuser2012a,
author = {Heuser, Thomas and Barber, Cynthia F. and Lin, Jianfeng and Krell, Jeremy and Rebesco, Matthew and Porter, Mary E. and Nicastro, Daniela},
title = {Cryoelectron tomography reveals doublet-specific structures and unique interactions in the I1 dynein.},
journal = {Proc Natl Acad Sci U S A},
school = {Biology Department, Rosenstiel Center, Brandeis University, Waltham, MA 02454, USA.},
year = {2012},
volume = {109},
number = {30},
pages = {E2067--E2076},
url = {http://dx.doi.org/10.1073/pnas.1120690109},
doi = {10.1073/pnas.1120690109}
}
|
| Heuser T, Dymek EE, Lin J, Smith EF and Nicastro D (2012), "The CSC connects three major axonemal complexes involved in dynein regulation.", Mol Biol Cell. Vol. 23(16), pp. 3143-3155. |
| Abstract: Motile cilia and flagella are highly conserved organelles that play important roles in human health and development. We recently discovered a calmodulin- and spoke-associ-ated complex (CSC) that is required for wild-type motility and for the stable assembly of a subset of radial spokes. Using cryo-electron tomography, we present the first structure-based localization model of the CSC. Chlamydomonas flagella have two full-length radial spokes, RS1 and RS2, and a shorter RS3 homologue, the RS3 stand-in (RS3S). Using newly developed techniques for analyzing samples with structural heterogeneity, we demonstrate that the CSC connects three major axonemal complexes involved in dynein regulation: RS2, the nexin-dynein regulatory complex (N-DRC), and RS3S. These results provide insights into how signals from the radial spokes may be transmitted to the N-DRC and ultimately to the dynein motors. Our results also indicate that although structurally very similar, RS1 and RS2 likely serve different functions in regulating flagellar motility. |
BibTeX:
@article{Heuser2012,
author = {Heuser, Thomas and Dymek, Erin E. and Lin, Jianfeng and Smith, Elizabeth F. and Nicastro, Daniela},
title = {The CSC connects three major axonemal complexes involved in dynein regulation.},
journal = {Mol Biol Cell},
school = {Biology Department, Rosenstiel Center, Brandeis University, Waltham, MA 02454, USA.},
year = {2012},
volume = {23},
number = {16},
pages = {3143--3155},
url = {http://dx.doi.org/10.1091/mbc.E12-05-0357},
doi = {10.1091/mbc.E12-05-0357}
}
|
| Heuser T, Raytchev M, Krell J, Porter ME and Nicastro D (2009), "The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella.", J Cell Biol. Vol. 187(6), pp. 921-933. |
| Abstract: Cilia and flagella are highly conserved microtubule (MT)-based organelles with motile and sensory functions, and ciliary defects have been linked to several human diseases. The 9 + 2 structure of motile axonemes contains nine MT doublets interconnected by nexin links, which surround a central pair of singlet MTs. Motility is generated by the orchestrated activity of thousands of dynein motors, which drive interdoublet sliding. A key regulator of motor activity is the dynein regulatory complex (DRC), but detailed structural information is lacking. Using cryoelectron tomography of wild-type and mutant axonemes from Chlamydomonas reinhardtii, we visualized the DRC in situ at molecular resolution. We present the three-dimensional structure of the DRC, including a model for its subunit organization and intermolecular connections that establish the DRC as a major regulatory node. We further demonstrate that the DRC is the nexin link, which is thought to be critical for the generation of axonemal bending. |
BibTeX:
@article{Heuser2009,
author = {Heuser, Thomas and Raytchev, Milen and Krell, Jeremy and Porter, Mary E. and Nicastro, Daniela},
title = {The dynein regulatory complex is the nexin link and a major regulatory node in cilia and flagella.},
journal = {J Cell Biol},
school = {Biology Department, Brandeis University, Waltham, MA 02453, USA.},
year = {2009},
volume = {187},
number = {6},
pages = {921--933},
url = {http://dx.doi.org/10.1083/jcb.200908067},
doi = {10.1083/jcb.200908067}
}
|
| Himes BA and Zhang P (2018), "emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging.", Nature methods. Vol. 15, pp. 955-961. |
| Abstract: Macromolecular complexes are intrinsically flexible and often challenging to purify for structure determination by single-particle cryo-electron microscopy (cryo-EM). Such complexes can be studied by cryo-electron tomography (cryo-ET) combined with subtomogram alignment and classification, which in exceptional cases achieves subnanometer resolution, yielding insight into structure-function relationships. However, it remains challenging to apply this approach to specimens that exhibit conformational or compositional heterogeneity or are present in low abundance. To address this, we developed emClarity ( https://github.com/bHimes/emClarity/wiki ), a GPU-accelerated image-processing package featuring an iterative tomographic tilt-series refinement algorithm that uses subtomograms as fiducial markers and a 3D-sampling-function-compensated, multi-scale principal component analysis classification method. We demonstrate that our approach offers substantial improvement in the resolution of maps and in the separation of different functional states of macromolecular complexes compared with current state-of-the-art software. |
BibTeX:
@article{Himes2018,
author = {Himes, Benjamin A and Zhang, Peijun},
title = {emClarity: software for high-resolution cryo-electron tomography and subtomogram averaging.},
journal = {Nature methods},
year = {2018},
volume = {15},
pages = {955--961},
doi = {10.1038/s41592-018-0167-z}
}
|
| Hoenger A (2014), "High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles.", Protoplasma. Vol. 251(2), pp. 417-427. |
| Abstract: Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ± 60 and ± 70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1-2° or less and a tilt range of ± 60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree. |
BibTeX:
@article{Hoenger2014,
author = {Hoenger, Andreas},
title = {High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles.},
journal = {Protoplasma},
school = {Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Boulder, CO, 80309, USA, hoenger@colorado.edu.},
year = {2014},
volume = {251},
number = {2},
pages = {417--427},
url = {http://dx.doi.org/10.1007/s00709-013-0600-1},
doi = {10.1007/s00709-013-0600-1}
}
|
| Hoenger A and McIntosh JR (2009), "Probing the macromolecular organization of cells by electron tomography.", Curr Opin Cell Biol. Vol. 21(1), pp. 89-96. |
| Abstract: A major goal in cell biology is to understand the functional organization of macromolecular complexes in vivo. Electron microscopy is helping cell biologists to achieve this goal, thanks to its ability to resolve structural details in the nanometer range. While issues related to specimen preparation, imaging, and image interpretation make this approach to cell architecture difficult, recent improvements in methods, equipment, and software have facilitated the study of both important macromolecular complexes and comparatively large volumes from cellular specimens. Here, we describe recent progress in electron microscopy of cells and the ways in which the relevant methodologies are helping to elucidate cell architecture. |
BibTeX:
@article{Hoenger2009,
author = {Hoenger, Andreas and McIntosh, J Richard},
title = {Probing the macromolecular organization of cells by electron tomography.},
journal = {Curr Opin Cell Biol},
school = {Boulder Laboratory for 3-Dimensional Electron Microscopy of Cells and Molecules, Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USA.},
year = {2009},
volume = {21},
number = {1},
pages = {89--96},
url = {http://dx.doi.org/10.1016/j.ceb.2008.12.003},
doi = {10.1016/j.ceb.2008.12.003}
}
|
| Hoffmann PC, Giandomenico SL, Ganeva I, Wozny MR, Sutcliffe M, Lancaster MA and Kukulski W (2021), "Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids.", eLife. Vol. 10 |
| Abstract: During brain development, axons must extend over great distances in a relatively short amount of time. How the subcellular architecture of the growing axon sustains the requirements for such rapid build-up of cellular constituents has remained elusive. Human axons have been particularly poorly accessible to imaging at high resolution in a near-native context. Here, we present a method that combines cryo-correlative light microscopy and electron tomography with human cerebral organoid technology to visualize growing axon tracts. Our data reveal a wealth of structural details on the arrangement of macromolecules, cytoskeletal components, and organelles in elongating axon shafts. In particular, the intricate shape of the endoplasmic reticulum is consistent with its role in fulfilling the high demand for lipid biosynthesis to support growth. Furthermore, the scarcity of ribosomes within the growing shaft suggests limited translational competence during expansion of this compartment. These findings establish our approach as a powerful resource for investigating the ultrastructure of defined neuronal compartments. |
BibTeX:
@article{Hoffmann2021,
author = {Hoffmann, Patrick C. and Giandomenico, Stefano L. and Ganeva, Iva and Wozny, Michael R. and Sutcliffe, Magdalena and Lancaster, Madeline A. and Kukulski, Wanda},
title = {Electron cryo-tomography reveals the subcellular architecture of growing axons in human brain organoids.},
journal = {eLife},
year = {2021},
volume = {10},
doi = {10.7554/eLife.70269}
}
|
| Höög JL, Bouchet-Marquis C, McIntosh JR, Hoenger A and Gull K (2012), "Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei.", J Struct Biol. Vol. 178(2), pp. 189-198. |
| Abstract: Trypanosoma brucei is a uni-cellular protist that causes African sleeping sickness. These parasites have a flagellum that is attached to the cell body and is indispensible for its motility. The flagellum consists of a canonical 9+2 axoneme and a paraflagellar rod (PFR), an intricate tripartite, fibrous structure that is connected to the axoneme. In this paper we describe results from cryo-electron tomography of unperturbed flagella. This method revealed novel structures that are likely involved in attaching the flagellum to the cell. We also show the first cryo-electron tomographic images of a basal body in situ, revealing electron dense structures inside its triplet microtubules. Sub-tomogram averaging of the PFR revealed that its distal region is organized as an orthorhombic crystal. |
BibTeX:
@article{Hoeoeg2012,
author = {Höög, Johanna L. and Bouchet-Marquis, Cédric and McIntosh, J Richard and Hoenger, Andreas and Gull, Keith},
title = {Cryo-electron tomography and 3-D analysis of the intact flagellum in Trypanosoma brucei.},
journal = {J Struct Biol},
school = {The Boulder Laboratory for 3-D Electron Microscopy of Cells, MCD-Biology, University of Colorado at Boulder, Boulder, CO 80309-0347, USA. hoog@colorado.edu},
year = {2012},
volume = {178},
number = {2},
pages = {189--198},
url = {http://dx.doi.org/10.1016/j.jsb.2012.01.009},
doi = {10.1016/j.jsb.2012.01.009}
}
|
| Hover S, Charlton FW, Hellert J, Barr JN, Mankouri J and Fontana J (), "Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K+ during entry". |
BibTeX:
@misc{Hover,
author = {Samantha Hover and Frank W. Charlton and Jan Hellert and John N. Barr and Jamel Mankouri and Juan Fontana},
title = {Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K+ during entry},
doi = {10.1101/2022.08.11.503604}
}
|
| Hu H, Wu X, Wang H, Wang H and Zhou J (2019), "Photo-reduction of Ag nanoparticles by using cellulose-based micelles as soft templates: Catalytic and antimicrobial activities", Carbohydrate polymers. Vol. 213, pp. 419-427. Elsevier.
[BibTeX] |
BibTeX:
@article{Hu2019,
author = {Hu, Haoze and Wu, Xiaoqing and Wang, Haoying and Wang, Hongyu and Zhou, Jinping},
title = {Photo-reduction of Ag nanoparticles by using cellulose-based micelles as soft templates: Catalytic and antimicrobial activities},
journal = {Carbohydrate polymers},
publisher = {Elsevier},
year = {2019},
volume = {213},
pages = {419--427}
}
|
| Hunter EL, Lechtreck K, Fu G, Hwang J, Lin H, Gokhale A, Alford LM, Lewis B, Yamamoto R, Kamiya R, Yang F, Nicastro D, Dutcher SK, Wirschell M and Sale WS (2018), "The IDA3 adapter, required for intraflagellar transport of I1 dynein, is regulated by ciliary length.", Molecular biology of the cell. Vol. 29, pp. 886-896. |
| Abstract: We determined how the ciliary motor I1 dynein is transported. A specialized adapter, IDA3, facilitates I1 dynein attachment to the ciliary transporter called intraflagellar transport (IFT). Loading of IDA3 and I1 dynein on IFT is regulated by ciliary length. |
BibTeX:
@article{Hunter2018,
author = {Hunter, Emily L and Lechtreck, Karl and Fu, Gang and Hwang, Juyeon and Lin, Huawen and Gokhale, Avanti and Alford, Lea M and Lewis, Brian and Yamamoto, Ryosuke and Kamiya, Ritsu and Yang, Fan and Nicastro, Daniela and Dutcher, Susan K and Wirschell, Maureen and Sale, Winfield S},
title = {The IDA3 adapter, required for intraflagellar transport of I1 dynein, is regulated by ciliary length.},
journal = {Molecular biology of the cell},
year = {2018},
volume = {29},
pages = {886--896},
doi = {10.1091/mbc.E17-12-0729}
}
|
| Imhof S, Zhang J, Wang H, Bui KH, Nguyen H, Atanasov I, Hui WH, Yang SK, Zhou ZH and Hill KL (2019), "Cryo electron tomography with Volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen , javax.xml.bind.JAXBElement@57ceca02, .", eLife. Vol. 8 |
| Abstract: The 96-nm axonemal repeat includes dynein motors and accessory structures as the foundation for motility of eukaryotic flagella and cilia. However, high-resolution 3D axoneme structures are unavailable for organisms among the Excavates, which include pathogens of medical and economic importance. Here we report cryo electron tomography structure of the 96-nm repeat from , a protozoan parasite in the Excavate lineage that causes African trypanosomiasis. We examined bloodstream and procyclic life cycle stages, and a knockdown lacking DRC11/CMF22 of the nexin dynein regulatory complex (NDRC). Sub-tomogram averaging yields a resolution of 21.8 Å for the 96-nm repeat. We discovered several lineage-specific structures, including novel inter-doublet linkages and microtubule inner proteins (MIPs). We establish that DRC11/CMF22 is required for the NDRC proximal lobe that binds the adjacent doublet microtubule. We propose that lineage-specific elaboration of axoneme structure in reflects adaptations to support unique motility needs in diverse host environments. |
BibTeX:
@article{Imhof2019,
author = {Imhof, Simon and Zhang, Jiayan and Wang, Hui and Bui, Khanh Huy and Nguyen, Hoangkim and Atanasov, Ivo and Hui, Wong H and Yang, Shun Kai and Zhou, Z Hong and Hill, Kent L},
title = {Cryo electron tomography with Volta phase plate reveals novel structural foundations of the 96-nm axonemal repeat in the pathogen , javax.xml.bind.JAXBElement@57ceca02, .},
journal = {eLife},
year = {2019},
volume = {8},
doi = {10.7554/eLife.52058}
}
|
| Jordan MA, Diener DR, Stepanek L and Pigino G (2018), "The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterograde movement in cilia.", Nature cell biology. Vol. 20, pp. 1250-1255. |
| Abstract: Movement of cargos along microtubules plays key roles in diverse cellular processes, from signalling to mitosis. In cilia, rapid movement of ciliary components along the microtubules to and from the assembly site is essential for the assembly and disassembly of the structure itself . This bidirectional transport, known as intraflagellar transport (IFT) , is driven by the anterograde motor kinesin-2 and the retrograde motor dynein-1b (dynein-2 in mammals) . However, to drive retrograde transport, dynein-1b must first be delivered to the ciliary tip by anterograde IFT . Although, the presence of opposing motors in bidirectional transport processes often leads to periodic stalling and slowing of cargos , IFT is highly processive . Using cryo-electron tomography, we show that a tug-of-war between kinesin-2 and dynein-1b is prevented by loading dynein-1b onto anterograde IFT trains in an autoinhibited form and by positioning it away from the microtubule track to prevent binding. Once at the ciliary tip, dynein-1b must transition into an active form and engage microtubules to power retrograde trains. These findings provide a striking example of how coordinated structural changes mediate the behaviour of complex cellular machinery. |
BibTeX:
@article{Jordan2018,
author = {Jordan, Mareike A and Diener, Dennis R and Stepanek, Ludek and Pigino, Gaia},
title = {The cryo-EM structure of intraflagellar transport trains reveals how dynein is inactivated to ensure unidirectional anterograde movement in cilia.},
journal = {Nature cell biology},
year = {2018},
volume = {20},
pages = {1250--1255},
doi = {10.1038/s41556-018-0213-1}
}
|
| Jordan MA and Pigino G (2019), "In situ cryo-electron tomography and subtomogram averaging of intraflagellar transport trains.", Methods in cell biology. Vol. 152, pp. 179-195. |
| Abstract: In situ cryo-electron tomography (cryo-ET) and subtomogram averaging are powerful tools, able to provide 3D structures of biological samples at sub-nanometer resolution, while preserving information about cellular context and higher-order assembly. Best results are typically achieved, when applied to highly repetitive structures, such as viruses. Other typical examples are protein complexes that decorate long stretches along ciliary microtubules at stereotypical and precise repeats, such as axonemal dyneins. For such cases, a plethora of subtomogram averaging protocols exist. In this chapter, we show how we use cryo-ET and subtomogram averaging to study the architecture of the intraflagellar transport (IFT) machinery, a more challenging target that appears only in low copy numbers per tomogram. In the IFT trains, repeating units of IFT adaptor proteins engage two oppositely directed molecular motors to quickly shuttle ciliary building blocks and other proteins to the tip of the cilium and/or back to the base. This dynamic and sporadic nature of IFT trains poses challenges for determining the localization or precise orientation of the particles to be averaged. Solutions to these problems are described in this chapter. |
BibTeX:
@article{Jordan2019,
author = {Jordan, Mareike A and Pigino, Gaia},
title = {In situ cryo-electron tomography and subtomogram averaging of intraflagellar transport trains.},
journal = {Methods in cell biology},
year = {2019},
volume = {152},
pages = {179--195},
doi = {10.1016/bs.mcb.2019.04.005}
}
|
| Julkapli NM and Bagheri S (2018), "Surface Modification of Titania/Gold Nanoparticles for Photocatalytic Applications", In Nanocatalysts in Environmental Applications., pp. 25-35. Springer.
[BibTeX] |
BibTeX:
@incollection{Julkapli2018,
author = {Julkapli, Nurhidayatullaili Muhd and Bagheri, Samira},
title = {Surface Modification of Titania/Gold Nanoparticles for Photocatalytic Applications},
booktitle = {Nanocatalysts in Environmental Applications},
publisher = {Springer},
year = {2018},
pages = {25--35}
}
|
| Julkapli NM, Bagheri S and Abdullah FZ (2017), "Photocatalytic activities and photoinduced fusion of gold-modified titania nanoparticle", Reviews in Inorganic Chemistry. Vol. 37(2), pp. 95-103. De Gruyter.
[BibTeX] |
BibTeX:
@article{Julkapli2017,
author = {Julkapli, Nurhidayatullaili Muhd and Bagheri, Samira and Abdullah, Fatimah Zahara},
title = {Photocatalytic activities and photoinduced fusion of gold-modified titania nanoparticle},
journal = {Reviews in Inorganic Chemistry},
publisher = {De Gruyter},
year = {2017},
volume = {37},
number = {2},
pages = {95--103}
}
|
| Kaplan M, Ghosal D, Subramanian P, Oikonomou CM, Kjaer A, Pirbadian S, Ortega DR, Briegel A, El-Naggar MY and Jensen GJ (2019), "The presence and absence of periplasmic rings in bacterial flagellar motors correlates with stator type.", eLife. Vol. 8 |
| Abstract: The bacterial flagellar motor, a cell-envelope-embedded macromolecular machine that functions as a cellular propeller, exhibits significant structural variability between species. Different torque-generating stator modules allow motors to operate in different pH, salt or viscosity levels. How such diversity evolved is unknown. Here, we use electron cryo-tomography to determine the in situ macromolecular structures of three Gammaproteobacteria motors: , , and , providing the first views of intact motors with dual stator systems. Complementing our imaging with bioinformatics analysis, we find a correlation between the motor's stator system and its structural elaboration. Motors with a single H -driven stator have only the core periplasmic P- and L-rings; those with dual H -driven stators have an elaborated P-ring; and motors with Na or Na /H -driven stators have both their P- and L-rings embellished. Our results suggest an evolution of structural elaboration that may have enabled pathogenic bacteria to colonize higher-viscosity environments in animal hosts. |
BibTeX:
@article{Kaplan2019,
author = {Kaplan, Mohammed and Ghosal, Debnath and Subramanian, Poorna and Oikonomou, Catherine M and Kjaer, Andreas and Pirbadian, Sahand and Ortega, Davi R and Briegel, Ariane and El-Naggar, Mohamed Y and Jensen, Grant J},
title = {The presence and absence of periplasmic rings in bacterial flagellar motors correlates with stator type.},
journal = {eLife},
year = {2019},
volume = {8},
doi = {10.7554/eLife.43487}
}
|
| Kaplan M, Nicolas WJ, Zhao W, Carter SD, Metskas LA, Chreifi G, Ghosal D and Jensen GJ (2021), "In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography.", Methods in molecular biology (Clifton, N.J.). Vol. 2215, pp. 83-111. |
| Abstract: Electron cryo-tomography (cryo-ET) is a technique that allows the investigation of intact macromolecular complexes while they are in their cellular milieu. Over the years, cryo-ET has had a huge impact on our understanding of how large biomolecular complexes look like, how they assemble, disassemble, function, and evolve(d). Recent hardware and software developments and combining cryo-ET with other techniques, e.g., focused ion beam milling (FIB-milling) and cryo-light microscopy, has extended the realm of cryo-ET to include transient molecular complexes embedded deep in thick samples (like eukaryotic cells) and enhanced the resolution of structures obtained by cryo-ET. In this chapter, we will present an outline of how to perform cryo-ET studies on a wide variety of biological samples including prokaryotic and eukaryotic cells and biological plant tissues. This outline will include sample preparation, data collection, and data processing as well as hybrid approaches like FIB-milling, cryosectioning, and cryo-correlated light and electron microscopy (cryo-CLEM). |
BibTeX:
@article{Kaplan2021,
author = {Kaplan, Mohammed and Nicolas, William J. and Zhao, Wei and Carter, Stephen D. and Metskas, Lauren Ann and Chreifi, Georges and Ghosal, Debnath and Jensen, Grant J.},
title = {In Situ Imaging and Structure Determination of Biomolecular Complexes Using Electron Cryo-Tomography.},
journal = {Methods in molecular biology (Clifton, N.J.)},
year = {2021},
volume = {2215},
pages = {83--111},
doi = {10.1007/978-1-0716-0966-8_4}
}
|
| Kaplan M, Oikonomou CM, Wood CR, Chreifi G, Ghosal D, Dobro MJ, Yao Q, Pal RR, Baidya AK, Liu Y, Maggi S, McDowall AW, Ben-Yehuda S, Rosenshine I, Briegel A, Beeby M, Chang Y-W, Shaffer CL and Jensen GJ (2022), "Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System" Vol. 204 |
BibTeX:
@article{Kaplan2022a,
author = {Mohammed Kaplan and Catherine M. Oikonomou and Cecily R. Wood and Georges Chreifi and Debnath Ghosal and Megan J. Dobro and Qing Yao and Ritesh Ranjan Pal and Amit K. Baidya and Yuxi Liu and Stefano Maggi and Alasdair W. McDowall and Sigal Ben-Yehuda and Ilan Rosenshine and Ariane Briegel and Morgan Beeby and Yi-Wei Chang and Carrie L. Shaffer and Grant J. Jensen},
title = {Discovery of a Novel Inner Membrane-Associated Bacterial Structure Related to the Flagellar Type III Secretion System},
year = {2022},
volume = {204},
doi = {10.1128/jb.00144-22}
}
|
| Kaplan M, Oikonomou CM, Wood CR, Chreifi G, Subramanian P, Ortega DR, Chang Y, Beeby M, Shaffer CL and Jensen GJ (2022), "Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors" Vol. 41 |
BibTeX:
@article{Kaplan2022,
author = {Mohammed Kaplan and Catherine M. Oikonomou and Cecily R. Wood and Georges Chreifi and Poorna Subramanian and Davi R. Ortega and Yi‐Wei Chang and Morgan Beeby and Carrie L. Shaffer and Grant J. Jensen},
title = {Novel transient cytoplasmic rings stabilize assembling bacterial flagellar motors},
year = {2022},
volume = {41},
doi = {10.15252/embj.2021109523}
}
|
| Kaplan M, Subramanian P, Ghosal D, Oikonomou CM, Pirbadian S, Starwalt-Lee R, Mageswaran SK, Ortega DR, Gralnick JA, El-Naggar MY and Jensen GJ (2019), "In situ imaging of the bacterial flagellar motor disassembly and assembly processes.", The EMBO journal. Vol. 38, pp. e100957. |
| Abstract: The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process. |
BibTeX:
@article{Kaplan2019a,
author = {Kaplan, Mohammed and Subramanian, Poorna and Ghosal, Debnath and Oikonomou, Catherine M and Pirbadian, Sahand and Starwalt-Lee, Ruth and Mageswaran, Shrawan Kumar and Ortega, Davi R and Gralnick, Jeffrey A and El-Naggar, Mohamed Y and Jensen, Grant J},
title = {In situ imaging of the bacterial flagellar motor disassembly and assembly processes.},
journal = {The EMBO journal},
year = {2019},
volume = {38},
pages = {e100957},
doi = {10.15252/embj.2018100957}
}
|
| Ke Z, Strauss JD, Hampton CM, Brindley MA, Dillard RS, Leon F, Lamb KM, Plemper RK and Wright ER (2018), "Promotion of virus assembly and organization by the measles virus matrix protein.", Nature communications. Vol. 9, pp. 1736. |
| Abstract: Measles virus (MeV) remains a major human pathogen, but there are presently no licensed antivirals to treat MeV or other paramyxoviruses. Here, we use cryo-electron tomography (cryo-ET) to elucidate the principles governing paramyxovirus assembly in MeV-infected human cells. The three-dimensional (3D) arrangement of the MeV structural proteins including the surface glycoproteins (F and H), matrix protein (M), and the ribonucleoprotein complex (RNP) are characterized at stages of virus assembly and budding, and in released virus particles. The M protein is observed as an organized two-dimensional (2D) paracrystalline array associated with the membrane. A two-layered F-M lattice is revealed suggesting that interactions between F and M may coordinate processes essential for MeV assembly. The RNP complex remains associated with and in close proximity to the M lattice. In this model, the M lattice facilitates the well-ordered incorporation and concentration of the surface glycoproteins and the RNP at sites of virus assembly. |
BibTeX:
@article{Ke2018,
author = {Ke, Zunlong and Strauss, Joshua D and Hampton, Cheri M and Brindley, Melinda A and Dillard, Rebecca S and Leon, Fredrick and Lamb, Kristen M and Plemper, Richard K and Wright, Elizabeth R},
title = {Promotion of virus assembly and organization by the measles virus matrix protein.},
journal = {Nature communications},
year = {2018},
volume = {9},
pages = {1736},
doi = {10.1038/s41467-018-04058-2}
}
|
| Kiesel P, Alvarez Viar G, Tsoy N, Maraspini R, Gorilak P, Varga V, Honigmann A and Pigino G (2020), "The molecular structure of mammalian primary cilia revealed by cryo-electron tomography.", Nature structural & molecular biology. Vol. 27, pp. 1115-1124. |
| Abstract: Primary cilia are microtubule-based organelles that are important for signaling and sensing in eukaryotic cells. Unlike the thoroughly studied motile cilia, the three-dimensional architecture and molecular composition of primary cilia are largely unexplored. Yet, studying these aspects is necessary to understand how primary cilia function in health and disease. We developed an enabling method for investigating the structure of primary cilia isolated from MDCK-II cells at molecular resolution by cryo-electron tomography. We show that the textbook '9 + 0' arrangement of microtubule doublets is only present at the primary cilium base. A few microns out, the architecture changes into an unstructured bundle of EB1-decorated microtubules and actin filaments, putting an end to a long debate on the presence or absence of actin filaments in primary cilia. Our work provides a plethora of insights into the molecular structure of primary cilia and offers a methodological framework to study these important organelles. |
BibTeX:
@article{Kiesel2020,
author = {Kiesel, Petra and Alvarez Viar, Gonzalo and Tsoy, Nikolai and Maraspini, Riccardo and Gorilak, Peter and Varga, Vladimir and Honigmann, Alf and Pigino, Gaia},
title = {The molecular structure of mammalian primary cilia revealed by cryo-electron tomography.},
journal = {Nature structural & molecular biology},
year = {2020},
volume = {27},
pages = {1115--1124},
doi = {10.1038/s41594-020-0507-4}
}
|
| Kirmse R, Bouchet-Marquis C, Page C and Hoenger A (2010), "Three-dimensional cryo-electron microscopy on intermediate filaments.", Methods Cell Biol. Vol. 96, pp. 565-589. |
| Abstract: Together with microtubules and actin filaments (F-actin), intermediate filaments (IFs) form the cytoskeleton of metazoan cells. However, unlike the other two entities that are extremely conserved, IFs are much more diverse and are grouped into five different families. In contrast to microtubules and F-actin, IFs do not exhibit a polarity, which may be the reason that no molecular motors travel along them. The molecular structure of IFs is less well resolved than that of the other cytoskeletal systems. This is partially due to their functional variability, tissue-specific expression, and their intrinsic structural properties. IFs are composed mostly of relatively smooth protofibrils formed by antiparallel arranged α-helical coiled-coil bundles flanked by small globular domains at either end. These features make them difficult to study by various electron microscopy methods or atomic force microscopy (AFM). Furthermore, the elongated shape of monomeric or dimeric IF units interferes with the formation of highly ordered three-dimensional (3-D) crystals suitable for atomic resolution crystallography. So far, most of the data we currently have on IF macromolecular structures come from electron microscopy of negatively stained samples, and fragmented α-helical coiled-coil units solved by X-ray diffraction. In addition, AFM allows the observation of the dynamic states of IFs in solution and delivers a new view into the assembly properties of IFs. Here, we discuss the applicability of cryo-electron microscopy (cryo-EM) and cryo-electron tomography (cryo-ET) for the field. Both methods are strongly related and have only recently been applied to IFs. However, cryo-EM revealed distinct new features within IFs that have not been seen before, and cryo-ET adds a 3-D view of IFs revealing the path and number of protofilaments within the various IF assemblies. |
BibTeX:
@article{Kirmse2010,
author = {Kirmse, Robert and Bouchet-Marquis, Cédric and Page, Cynthia and Hoenger, Andreas},
title = {Three-dimensional cryo-electron microscopy on intermediate filaments.},
journal = {Methods Cell Biol},
school = {The Boulder Laboratory for 3-D Microscopy of Cells, University of Colorado at Boulder, Boulder, Colorado 80309-0347, USA.},
year = {2010},
volume = {96},
pages = {565--589},
url = {http://dx.doi.org/10.1016/S0091-679X(10)96023-8},
doi = {10.1016/S0091-679X(10)96023-8}
}
|
| Knowles MR, Ostrowski LE, Loges NT, Hurd T, Leigh MW, Huang L, Wolf WE, Carson JL, Hazucha MJ, Yin W, Davis SD, Dell SD, Ferkol TW, Sagel SD, Olivier KN, Jahnke C, Olbrich H, Werner C, Raidt J, Wallmeier J, Pennekamp P, Dougherty GW, Hjeij R, Gee HY, Otto EA, Halbritter J, Chaki M, Diaz KA, Braun DA, Porath JD, Schueler M, Baktai G, Griese M, Turner EH, Lewis AP, Bamshad MJ, Nickerson DA, Hildebrandt F, Shendure J, Omran H and Zariwala MA (2013), "Mutations in SPAG1 cause primary ciliary dyskinesia associated with defective outer and inner dynein arms.", American journal of human genetics. Vol. 93, pp. 711-720. |
| Abstract: Primary ciliary dyskinesia (PCD) is a genetically heterogeneous, autosomal-recessive disorder, characterized by oto-sino-pulmonary disease and situs abnormalities. PCD-causing mutations have been identified in 20 genes, but collectively they account for only ∼65% of all PCDs. To identify mutations in additional genes that cause PCD, we performed exome sequencing on three unrelated probands with ciliary outer and inner dynein arm (ODA+IDA) defects. Mutations in SPAG1 were identified in one family with three affected siblings. Further screening of SPAG1 in 98 unrelated affected individuals (62 with ODA+IDA defects, 35 with ODA defects, 1 without available ciliary ultrastructure) revealed biallelic loss-of-function mutations in 11 additional individuals (including one sib-pair). All 14 affected individuals with SPAG1 mutations had a characteristic PCD phenotype, including 8 with situs abnormalities. Additionally, all individuals with mutations who had defined ciliary ultrastructure had ODA+IDA defects. SPAG1 was present in human airway epithelial cell lysates but was not present in isolated axonemes, and immunofluorescence staining showed an absence of ODA and IDA proteins in cilia from an affected individual, thus indicating that SPAG1 probably plays a role in the cytoplasmic assembly and/or trafficking of the axonemal dynein arms. Zebrafish morpholino studies of spag1 produced cilia-related phenotypes previously reported for PCD-causing mutations in genes encoding cytoplasmic proteins. Together, these results demonstrate that mutations in SPAG1 cause PCD with ciliary ODA+IDA defects and that exome sequencing is useful to identify genetic causes of heterogeneous recessive disorders. |
BibTeX:
@article{Knowles2013,
author = {Knowles, Michael R. and Ostrowski, Lawrence E. and Loges, Niki T. and Hurd, Toby and Leigh, Margaret W. and Huang, Lu and Wolf, Whitney E. and Carson, Johnny L. and Hazucha, Milan J. and Yin, Weining and Davis, Stephanie D. and Dell, Sharon D. and Ferkol, Thomas W. and Sagel, Scott D. and Olivier, Kenneth N. and Jahnke, Charlotte and Olbrich, Heike and Werner, Claudius and Raidt, Johanna and Wallmeier, Julia and Pennekamp, Petra and Dougherty, Gerard W. and Hjeij, Rim and Gee, Heon Yung and Otto, Edgar A. and Halbritter, Jan and Chaki, Moumita and Diaz, Katrina A. and Braun, Daniela A. and Porath, Jonathan D. and Schueler, Markus and Baktai, György and Griese, Matthias and Turner, Emily H. and Lewis, Alexandra P. and Bamshad, Michael J. and Nickerson, Deborah A. and Hildebrandt, Friedhelm and Shendure, Jay and Omran, Heymut and Zariwala, Maimoona A.},
title = {Mutations in SPAG1 cause primary ciliary dyskinesia associated with defective outer and inner dynein arms.},
journal = {American journal of human genetics},
year = {2013},
volume = {93},
pages = {711--720},
doi = {10.1016/j.ajhg.2013.07.025}
}
|
| Kobayashi W, Hosoya N, Machida S, Miyagawa K and Kurumizaka H (2017), "SYCP3 regulates strand invasion activities of RAD51 and DMC1.", Genes to cells : devoted to molecular & cellular mechanisms. Vol. 22, pp. 799-809. |
| Abstract: The synaptonemal complex is a higher-ordered proteinaceous architecture formed between homologous chromosomes. SYCP3 is a major component of the lateral/axial elements in the synaptonemal complex and is essential for meiotic recombination. Previous genetic studies showed that SYCP3 functions in meiotic homologous recombination biased to interhomologous chromosomes, by regulating the strand invasion activities of the RAD51 and DMC1 recombinases. However, the mechanism by which SYCP3 regulates RAD51- and DMC1-mediated strand invasion remains elusive. In this study, we found that SYCP3 significantly suppresses the RAD51-mediated, but not the DMC1-mediated, strand invasion reaction by competing with HOP2-MND1, which is an activator for both RAD51 and DMC1. A SYCP3 mutant with defective RAD51 binding does not inhibit the RAD51-mediated homologous recombination in human cells. Therefore, SYCP3 may promote the DMC1-driven homologous recombination by attenuating the RAD51 activity during meiosis. |
BibTeX:
@article{Kobayashi2017,
author = {Kobayashi, Wataru and Hosoya, Noriko and Machida, Shinichi and Miyagawa, Kiyoshi and Kurumizaka, Hitoshi},
title = {SYCP3 regulates strand invasion activities of RAD51 and DMC1.},
journal = {Genes to cells : devoted to molecular & cellular mechanisms},
year = {2017},
volume = {22},
pages = {799--809},
doi = {10.1111/gtc.12513}
}
|
| Kornberg RD (2007), "The molecular basis of eukaryotic transcription", Proceedings of the National Academy of Sciences. Vol. 104(32), pp. 12955-12961. National Acad Sciences.
[BibTeX] |
BibTeX:
@article{Kornberg2007,
author = {Kornberg, Roger D},
title = {The molecular basis of eukaryotic transcription},
journal = {Proceedings of the National Academy of Sciences},
publisher = {National Acad Sciences},
year = {2007},
volume = {104},
number = {32},
pages = {12955--12961}
}
|
| Kornberg RD (2008), "The Challenge of Quasi-Regular Structures in Biology", In Physical Biology: From Atoms to Medicine., pp. 137-143. World Scientific.
[BibTeX] |
BibTeX:
@incollection{Kornberg2008,
author = {Kornberg, Roger D},
title = {The Challenge of Quasi-Regular Structures in Biology},
booktitle = {Physical Biology: From Atoms to Medicine},
publisher = {World Scientific},
year = {2008},
pages = {137--143}
}
|
| Kováčik L, Kereïche S, Matula P and Raška I (2014), "Sub-volume averaging of repetitive structural features in angularly filtered electron tomographic reconstructions.", Folia Biol (Praha). Vol. 60 Suppl 1, pp. 66-70. |
| Abstract: Electron tomographic reconstructions suffer from a number of artefacts arising from effects accompanying the processes of acquisition of a set of tilted projections of the specimen in a transmission electron microscope and from its subsequent computational handling. The most pronounced artefacts usually come from imprecise projection alignment, distortion of specimens during tomogram acquisition and from the presence of a region of missing data in the Fourier space, the "missing wedge". The ray artefacts caused by the presence of the missing wedge can be attenuated by the angular image filter, which attenuates the transition between the data and the missing wedge regions. In this work, we present an analysis of the influence of angular filtering on the resolution of averaged repetitive structural motives extracted from three-dimensional reconstructions of tomograms acquired in the single-axis tilting geometry. |
BibTeX:
@article{Kovacik2014,
author = {Kováčik, L. and Kereïche, S. and Matula, P. and Raška, I.},
title = {Sub-volume averaging of repetitive structural features in angularly filtered electron tomographic reconstructions.},
journal = {Folia Biol (Praha)},
school = {Institute of Cellular Biology and Pathology, First Faculty of Medicine, Charles University in Prague, Czech Republic.},
year = {2014},
volume = {60 Suppl 1},
pages = {66--70}
}
|
| Kubo T, Hou Y, Cochran DA, Witman GB and Oda T (2018), "A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1).", Molecular biology of the cell. Vol. 29, pp. 1060-1074. |
| Abstract: Motility of cilia/flagella is generated by a coordinated activity of thousands of dyneins. Inner dynein arms (IDAs) are particularly important for the formation of ciliary/flagellar waveforms, but the molecular mechanism of IDA regulation is poorly understood. Here we show using cryoelectron tomography and biochemical analyses of Chlamydomonas flagella that a conserved protein FAP44 forms a complex that tethers IDA f (I1 dynein) head domains to the A-tubule of the axonemal outer doublet microtubule. In wild-type flagella, IDA f showed little nucleotide-dependent movement except for a tilt in the f β head perpendicular to the microtubule-sliding direction. In the absence of the tether complex, however, addition of ATP and vanadate caused a large conformational change in the IDA f head domains, suggesting that the movement of IDA f is mechanically restricted by the tether complex. Motility defects in flagella missing the tether demonstrates the importance of the IDA f-tether interaction in the regulation of ciliary/flagellar beating. |
BibTeX:
@article{Kubo2018,
author = {Kubo, Tomohiro and Hou, Yuqing and Cochran, Deborah A and Witman, George B and Oda, Toshiyuki},
title = {A microtubule-dynein tethering complex regulates the axonemal inner dynein f (I1).},
journal = {Molecular biology of the cell},
year = {2018},
volume = {29},
pages = {1060--1074},
doi = {10.1091/mbc.E17-11-0689}
}
|
| Kubo T and Oda T (2017), "Electrostatic interaction between polyglutamylated tubulin and the nexin-dynein regulatory complex regulates flagellar motility.", Molecular biology of the cell. Vol. 28, pp. 2260-2266. |
| Abstract: Tubulins undergo various posttranslational modifications. Among them, polyglutamylation is involved in the motility of eukaryotic flagella and the stability of the axonemal microtubules. However, it remains unclear where polyglutamylated tubulin localizes precisely within the axoneme and how tubulin polyglutamylation affects flagellar motility. In this study, we identified the three-dimensional localization of the polyglutamylated tubulin in Chlamydomonas flagella using antibody labeling and cryo-electron tomography. Polyglutamylated tubulins specifically located in close proximity to a microtubule-cross-bridging structure called the nexin-dynein regulatory complex (N-DRC). Because N-DRC is positively charged, we hypothesized that there is an electrostatic interaction between the polyglutamylated tubulin and the N-DRC, and therefore we mutated the amino acid sequences of DRC4 to modify the charge of the N-DRC. We found that both augmentation and reduction of the positive charge on DRC4 resulted in reduced flagellar motility. Moreover, reduced motility in a mutant with a structurally defective N-DRC was partially restored by increasing the positive charge on DRC4. These results clearly indicate that beating motion of flagella is maintained by the electrostatic cross-bridge formed between the negatively charged polyglutamylated tubulins and the positively charged N-DRC. |
BibTeX:
@article{Kubo2017,
author = {Kubo, Tomohiro and Oda, Toshiyuki},
title = {Electrostatic interaction between polyglutamylated tubulin and the nexin-dynein regulatory complex regulates flagellar motility.},
journal = {Molecular biology of the cell},
year = {2017},
volume = {28},
pages = {2260--2266},
doi = {10.1091/mbc.E17-05-0285}
}
|
| Leigh KE, Navarro PP, Scaramuzza S, Chen W, Zhang Y, Castaño-Díez D and Kudryashev M (2019), "Subtomogram averaging from cryo-electron tomograms.", Methods in cell biology. Vol. 152, pp. 217-259. |
| Abstract: Cryo-electron tomography (cryo-ET) allows three-dimensional (3D) visualization of frozen-hydrated biological samples, such as protein complexes and cell organelles, in near-native environments at nanometer scale. Protein complexes that are present in multiple copies in a set of tomograms can be extracted, mutually aligned, and averaged to yield a signal-enhanced 3D structure up to sub-nanometer or even near-atomic resolution. This technique, called subtomogram averaging (StA), is powered by improvements in EM hardware and image processing software. Importantly, StA provides unique biological insights into the structure and function of cellular machinery in close-to-native contexts. In this chapter, we describe the principles and key steps of StA. We briefly cover sample preparation and data collection with an emphasis on image processing procedures related to tomographic reconstruction, subtomogram alignment, averaging, and classification. We conclude by summarizing current limitations and future directions of this technique with a focus on high-resolution StA. |
BibTeX:
@article{Leigh2019,
author = {Leigh, Kendra E and Navarro, Paula P and Scaramuzza, Stefano and Chen, Wenbo and Zhang, Yingyi and Castaño-Díez, Daniel and Kudryashev, Misha},
title = {Subtomogram averaging from cryo-electron tomograms.},
journal = {Methods in cell biology},
year = {2019},
volume = {152},
pages = {217--259},
doi = {10.1016/bs.mcb.2019.04.003}
}
|
| Leung MR, Roelofs MC, Ravi RT, Maitan P, Henning H, Zhang M, Bromfield EG, Howes SC, Gadella BM, Bloomfield-Gadêlha H and Zeev-Ben-Mordehai T (2021), "The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility.", The EMBO journal. Vol. 40, pp. e107410. |
| Abstract: Motile cilia are molecular machines used by a myriad of eukaryotic cells to swim through fluid environments. However, available molecular structures represent only a handful of cell types, limiting our understanding of how cilia are modified to support motility in diverse media. Here, we use cryo-focused ion beam milling-enabled cryo-electron tomography to image sperm flagella from three mammalian species. We resolve in-cell structures of centrioles, axonemal doublets, central pair apparatus, and endpiece singlets, revealing novel protofilament-bridging microtubule inner proteins throughout the flagellum. We present native structures of the flagellar base, which is crucial for shaping the flagellar beat. We show that outer dense fibers are directly coupled to microtubule doublets in the principal piece but not in the midpiece. Thus, mammalian sperm flagella are ornamented across scales, from protofilament-bracing structures reinforcing microtubules at the nano-scale to accessory structures that impose micron-scale asymmetries on the entire assembly. Our structures provide vital foundations for linking molecular structure to ciliary motility and evolution. |
BibTeX:
@article{Leung2021,
author = {Leung, Miguel Ricardo and Roelofs, Marc C. and Ravi, Ravi Teja and Maitan, Paula and Henning, Heiko and Zhang, Min and Bromfield, Elizabeth G. and Howes, Stuart C. and Gadella, Bart M. and Bloomfield-Gadêlha, Hermes and Zeev-Ben-Mordehai, Tzviya},
title = {The multi-scale architecture of mammalian sperm flagella and implications for ciliary motility.},
journal = {The EMBO journal},
year = {2021},
volume = {40},
pages = {e107410},
doi = {10.15252/embj.2020107410}
}
|
| Li P-N, Herrmann J, Tolar BB, Poitevin F, Ramdasi R, Bargar JR, Stahl DA, Jensen GJ, Francis CA, Wakatsuki S and van den Bedem H (2018), "Nutrient transport suggests an evolutionary basis for charged archaeal surface layer proteins.", The ISME journal. Vol. 12, pp. 2389-2402. |
| Abstract: Surface layers (S-layers) are two-dimensional, proteinaceous, porous lattices that form the outermost cell envelope component of virtually all archaea and many bacteria. Despite exceptional sequence diversity, S-layer proteins (SLPs) share important characteristics such as their ability to form crystalline sheets punctuated with nano-scale pores, and their propensity for charged amino acids, leading to acidic or basic isoelectric points. However, the precise function of S-layers, or the role of charged SLPs and how they relate to cellular metabolism is unknown. Nano-scale lattices affect the diffusion behavior of low-concentration solutes, even if they are significantly smaller than the pore size. Here, we offer a rationale for charged S-layer proteins in the context of the structural evolution of S-layers. Using the ammonia-oxidizing archaea (AOA) as a model for S-layer geometry, and a 2D electrodiffusion reaction computational framework to simulate diffusion and consumption of the charged solute ammonium (NH ), we find that the characteristic length scales of nanoporous S-layers elevate the concentration of NH in the pseudo-periplasmic space. Our simulations suggest an evolutionary, mechanistic basis for S-layer charge and shed light on the unique ability of some AOA to oxidize ammonia in environments with nanomolar NH availability, with broad implications for comparisons of ecologically distinct populations. |
BibTeX:
@article{Li2018,
author = {Li, Po-Nan and Herrmann, Jonathan and Tolar, Bradley B and Poitevin, Frédéric and Ramdasi, Rasika and Bargar, John R and Stahl, David A and Jensen, Grant J and Francis, Christopher A and Wakatsuki, Soichi and van den Bedem, Henry},
title = {Nutrient transport suggests an evolutionary basis for charged archaeal surface layer proteins.},
journal = {The ISME journal},
year = {2018},
volume = {12},
pages = {2389--2402},
doi = {10.1038/s41396-018-0191-0}
}
|
| Li Y-L, Chandrasekaran V, Carter SD, Woodward CL, Christensen DE, Dryden KA, Pornillos O, Yeager M, Ganser-Pornillos BK, Jensen GJ and Sundquist WI (2016), "Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids.", eLife. Vol. 5 |
| Abstract: TRIM5 proteins are restriction factors that block retroviral infections by binding viral capsids and preventing reverse transcription. Capsid recognition is mediated by C-terminal domains on TRIM5α (SPRY) or TRIMCyp (cyclophilin A), which interact weakly with capsids. Efficient capsid recognition also requires the conserved N-terminal tripartite motifs (TRIM), which mediate oligomerization and create avidity effects. To characterize how TRIM5 proteins recognize viral capsids, we developed methods for isolating native recombinant TRIM5 proteins and purifying stable HIV-1 capsids. Biochemical and EM analyses revealed that TRIM5 proteins assembled into hexagonal nets, both alone and on capsid surfaces. These nets comprised open hexameric rings, with the SPRY domains centered on the edges and the B-box and RING domains at the vertices. Thus, the principles of hexagonal TRIM5 assembly and capsid pattern recognition are conserved across primates, allowing TRIM5 assemblies to maintain the conformational plasticity necessary to recognize divergent and pleomorphic retroviral capsids. |
BibTeX:
@article{Li2016,
author = {Li, Yen-Li and Chandrasekaran, Viswanathan and Carter, Stephen D and Woodward, Cora L and Christensen, Devin E and Dryden, Kelly A and Pornillos, Owen and Yeager, Mark and Ganser-Pornillos, Barbie K and Jensen, Grant J and Sundquist, Wesley I},
title = {Primate TRIM5 proteins form hexagonal nets on HIV-1 capsids.},
journal = {eLife},
year = {2016},
volume = {5},
doi = {10.7554/eLife.16269}
}
|
| Liljeroos L, Krzyzaniak MA, Helenius A and Butcher SJ (2013), "Architecture of respiratory syncytial virus revealed by electron cryotomography.", Proc Natl Acad Sci U S A. Vol. 110(27), pp. 11133-11138. |
| Abstract: Human respiratory syncytial virus is a human pathogen that causes severe infection of the respiratory tract. Current information about the structure of the virus and its interaction with host cells is limited. We carried out an electron cryotomographic characterization of cell culture-grown human respiratory syncytial virus to determine the architecture of the virion. The particles ranged from 100 nm to 1,000 nm in diameter and were spherical, filamentous, or a combination of the two. The filamentous morphology correlated with the presence of a cylindrical matrix protein layer linked to the inner leaflet of the viral envelope and with local ordering of the glycoprotein spikes. Recombinant viruses with only the fusion protein in their envelope showed that these glycoproteins were predominantly in the postfusion conformation, but some were also in the prefusion form. The ribonucleocapsids were left-handed, randomly oriented, and curved inside the virions. In filamentous particles, they were often adjacent to an intermediate layer of protein assigned to M2-1 (an envelope-associated protein known to mediate association of ribonucleocapsids with the matrix protein). Our results indicate important differences in structure between the Paramyxovirinae and Pneumovirinae subfamilies within the Paramyxoviridae, and provide fresh insights into host cell exit of a serious pathogen. |
BibTeX:
@article{Liljeroos2013,
author = {Liljeroos, Lassi and Krzyzaniak, Magdalena Anna and Helenius, Ari and Butcher, Sarah Jane},
title = {Architecture of respiratory syncytial virus revealed by electron cryotomography.},
journal = {Proc Natl Acad Sci U S A},
school = {Department of Biosciences and Institute of Biotechnology, University of Helsinki, FIN-00790, Helsinki, Finland.},
year = {2013},
volume = {110},
number = {27},
pages = {11133--11138},
url = {http://dx.doi.org/10.1073/pnas.1309070110},
doi = {10.1073/pnas.1309070110}
}
|
| Lin J, Heuser T, Carbajal-González BI, Song K and Nicastro D (2012), "The structural heterogeneity of radial spokes in cilia and flagella is conserved.", Cytoskeleton (Hoboken). Vol. 69(2), pp. 88-100. |
| Abstract: Radial spokes (RSs) are ubiquitous components of motile cilia and flagella and play an essential role in transmitting signals that regulate the activity of the dynein motors, and thus ciliary and flagellar motility. In some organisms, the 96 nm axonemal repeat unit contains only a pair of spokes, RS1 and RS2, while most organisms have spoke triplets with an additional spoke RS3. The spoke pairs in Chlamydomonas flagella have been well characterized, while spoke triplets have received less attention. Here, we used cryoelectron tomography and subtomogram averaging to visualize the three-dimensional structure of spoke triplets in Strongylocentrotus purpuratus (sea urchin) sperm flagella in unprecedented detail. Only small differences were observed between RS1 and RS2, but the structure of RS3 was surprisingly unique and structurally different from the other two spokes. We observed novel doublet specific features that connect RS2, RS3, and the nexin-dynein regulatory complex, three key ciliary and flagellar structures. The distribution of these doublet specific structures suggests that they could be important for establishing the asymmetry of dynein activity required for the oscillatory movement of cilia and flagella. Surprisingly, a comparison with other organisms demonstrated both that this considerable RS heterogeneity is conserved and that organisms with RS pairs contain the basal part of RS3. This conserved RS heterogeneity may also reflect functional differences between the spokes and their involvement in regulating ciliary and flagellar motility. |
BibTeX:
@article{Lin2012,
author = {Lin, Jianfeng and Heuser, Thomas and Carbajal-González, Blanca I. and Song, Kangkang and Nicastro, Daniela},
title = {The structural heterogeneity of radial spokes in cilia and flagella is conserved.},
journal = {Cytoskeleton (Hoboken)},
school = {Department of Biology, Rosenstiel Center, MS029, Brandeis University, Waltham, Massachusetts 02454-9110, USA.},
year = {2012},
volume = {69},
number = {2},
pages = {88--100},
url = {http://dx.doi.org/10.1002/cm.21000},
doi = {10.1002/cm.21000}
}
|
| Lin J, Heuser T, Song K, Fu X and Nicastro D (2012), "One of the nine doublet microtubules of eukaryotic flagella exhibits unique and partially conserved structures.", PLoS One. Vol. 7(10), pp. e46494. |
| Abstract: The axonemal core of motile cilia and flagella consists of nine doublet microtubules surrounding two central single microtubules. Attached to the doublets are thousands of dynein motors that produce sliding between neighboring doublets, which in turn causes flagellar bending. Although many structural features of the axoneme have been described, structures that are unique to specific doublets remain largely uncharacterized. These doublet-specific structures introduce asymmetry into the axoneme and are likely important for the spatial control of local microtubule sliding. Here, we used cryo-electron tomography and doublet-specific averaging to determine the 3D structures of individual doublets in the flagella of two evolutionarily distant organisms, the protist Chlamydomonas and the sea urchin Strongylocentrotus. We demonstrate that, in both organisms, one of the nine doublets exhibits unique structural features. Some of these features are highly conserved, such as the inter-doublet link i-SUB5-6, which connects this doublet to its neighbor with a periodicity of 96 nm. We also show that the previously described inter-doublet links attached to this doublet, the o-SUB5-6 in Strongylocentrotus and the proximal 1-2 bridge in Chlamydomonas, are likely not homologous features. The presence of inter-doublet links and reduction of dynein arms indicate that inter-doublet sliding of this unique doublet against its neighbor is limited, providing a rigid plane perpendicular to the flagellar bending plane. These doublet-specific features and the non-sliding nature of these connected doublets suggest a structural basis for the asymmetric distribution of dynein activity and inter-doublet sliding, resulting in quasi-planar waveforms typical of 9+2 cilia and flagella. |
BibTeX:
@article{Lin2012a,
author = {Lin, Jianfeng and Heuser, Thomas and Song, Kangkang and Fu, Xiaofeng and Nicastro, Daniela},
title = {One of the nine doublet microtubules of eukaryotic flagella exhibits unique and partially conserved structures.},
journal = {PLoS One},
school = {Biology Department, Rosenstiel Center, Brandeis University, Waltham, Massachusetts, United States of America.},
year = {2012},
volume = {7},
number = {10},
pages = {e46494},
url = {http://dx.doi.org/10.1371/journal.pone.0046494},
doi = {10.1371/journal.pone.0046494}
}
|
| Lin J, Le TV, Augspurger K, Tritschler D, Bower R, Fu G, Perrone C, O'Toole ET, Mills KV, Dymek E, Smith E, Nicastro D and Porter ME (2019), "FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia.", Molecular biology of the cell. Vol. 30, pp. 2659-2680. |
| Abstract: Ciliary motility depends on both the precise spatial organization of multiple dynein motors within the 96 nm axonemal repeat and the highly coordinated interactions between different dyneins and regulatory complexes located at the base of the radial spokes. Mutations in genes encoding cytoplasmic assembly factors, intraflagellar transport factors, docking proteins, dynein subunits, and associated regulatory proteins can all lead to defects in dynein assembly and ciliary motility. Significant progress has been made in the identification of dynein subunits and extrinsic factors required for preassembly of dynein complexes in the cytoplasm, but less is known about the docking factors that specify the unique binding sites for the different dynein isoforms on the surface of the doublet microtubules. We have used insertional mutagenesis to identify a new locus, , required for targeting the assembly of a subset of inner dynein arms (IDAs) to a specific location in the 96 nm repeat. encodes flagellar-associated polypeptide (FAP)57/WDR65, a highly conserved WD repeat, coiled coil domain protein. Using high resolution proteomic and structural approaches, we find that FAP57 forms a discrete complex. Cryo-electron tomography coupled with epitope tagging and gold labeling reveal that FAP57 forms an extended structure that interconnects multiple IDAs and regulatory complexes. |
BibTeX:
@article{Lin2019,
author = {Lin, Jianfeng and Le, Thuc Vy and Augspurger, Katherine and Tritschler, Douglas and Bower, Raqual and Fu, Gang and Perrone, Catherine and O'Toole, Eileen T and Mills, Kristyn VanderWaal and Dymek, Erin and Smith, Elizabeth and Nicastro, Daniela and Porter, Mary E},
title = {FAP57/WDR65 targets assembly of a subset of inner arm dyneins and connects to regulatory hubs in cilia.},
journal = {Molecular biology of the cell},
year = {2019},
volume = {30},
pages = {2659--2680},
doi = {10.1091/mbc.E19-07-0367}
}
|
| Lin J and Nicastro D (2018), "Asymmetric distribution and spatial switching of dynein activity generates ciliary motility.", Science (New York, N.Y.). Vol. 360 |
| Abstract: Motile cilia and flagella are essential, highly conserved organelles, and their motility is driven by the coordinated activities of multiple dynein isoforms. The prevailing "switch-point" hypothesis posits that dyneins are asymmetrically activated to drive flagellar bending. To test this model, we applied cryo-electron tomography to visualize activity states of individual dyneins relative to their locations along beating flagella of sea urchin sperm cells. As predicted, bending was generated by the asymmetric distribution of dynein activity on opposite sides of the flagellum. However, contrary to predictions, most dyneins were in their active state, and the smaller population of conformationally inactive dyneins switched flagellar sides relative to the bending direction. Thus, our data suggest a "switch-inhibition" mechanism in which force imbalance is generated by inhibiting, rather than activating, dyneins on alternating sides of the flagellum. |
BibTeX:
@article{Lin2018f,
author = {Lin, Jianfeng and Nicastro, Daniela},
title = {Asymmetric distribution and spatial switching of dynein activity generates ciliary motility.},
journal = {Science (New York, N.Y.)},
year = {2018},
volume = {360},
doi = {10.1126/science.aar1968}
}
|
| Lin J, Okada K, Raytchev M, Smith MC and Nicastro D (2014), "Structural mechanism of the dynein power stroke.", Nat Cell Biol. Vol. 16(5), pp. 479-485. |
| Abstract: Dyneins are large microtubule motor proteins required for mitosis, intracellular transport and ciliary and flagellar motility. They generate force through a power-stroke mechanism, which is an ATP-consuming cycle of pre- and post-power-stroke conformational changes that cause relative motion between different dynein domains. However, key structural details of dynein's force generation remain elusive. Here, using cryo-electron tomography of intact, active (that is, beating), rapidly frozen sea urchin sperm flagella, we determined the in situ three-dimensional structures of all domains of both pre- and post-power-stroke dynein, including the previously unresolved linker and stalk of pre-power-stroke dynein. Our results reveal that the rotation of the head relative to the linker is the key action in dynein movement, and that there are at least two distinct pre-power-stroke conformations: pre-I (microtubule-detached) and pre-II (microtubule-bound). We provide three-dimensional reconstructions of native dyneins in three conformational states, in situ, allowing us to propose a molecular model of the structural cycle underlying dynein movement. |
BibTeX:
@article{Lin2014a,
author = {Lin, Jianfeng and Okada, Kyoko and Raytchev, Milen and Smith, Maria C. and Nicastro, Daniela},
title = {Structural mechanism of the dynein power stroke.},
journal = {Nat Cell Biol},
school = {Biology Department and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham Massachusetts 02454-9110, USA.},
year = {2014},
volume = {16},
number = {5},
pages = {479--485},
url = {http://dx.doi.org/10.1038/ncb2939},
doi = {10.1038/ncb2939}
}
|
| Linck R, Fu X, Lin J, Ouch C, Schefter A, Steffen W, Warren P and Nicastro D (2014), "Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments.", J Biol Chem. Vol. 289(25), pp. 17427-17444. |
| Abstract: Cilia and flagella are conserved, motile, and sensory cell organelles involved in signal transduction and human disease. Their scaffold consists of a 9-fold array of remarkably stable doublet microtubules (DMTs), along which motor proteins transmit force for ciliary motility and intraflagellar transport. DMTs possess Ribbons of three to four hyper-stable protofilaments whose location, organization, and specialized functions have been elusive. We performed a comprehensive analysis of the distribution and structural arrangements of Ribbon proteins from sea urchin sperm flagella, using quantitative immunobiochemistry, proteomics, immuno-cryo-electron microscopy, and tomography. Isolated Ribbons contain acetylated α-tubulin, β-tubulin, conserved protein Rib45, >95% of the axonemal tektins, and >95% of the calcium-binding proteins, Rib74 and Rib85.5, whose human homologues are related to the cause of juvenile myoclonic epilepsy. DMTs contain only one type of Ribbon, corresponding to protofilaments A11-12-13-1 of the A-tubule. Rib74 and Rib85.5 are associated with the Ribbon in the lumen of the A-tubule. Ribbons contain a single ∼5-nm wide filament, composed of equimolar tektins A, B, and C, which interact with the nexin-dynein regulatory complex. A summary of findings is presented, and the functions of Ribbon proteins are discussed in terms of the assembly and stability of DMTs, ciliary motility, and other microtubule systems. |
BibTeX:
@article{Linck2014,
author = {Linck, Richard and Fu, Xiaofeng and Lin, Jianfeng and Ouch, Christna and Schefter, Alexandra and Steffen, Walter and Warren, Peter and Nicastro, Daniela},
title = {Insights into the structure and function of ciliary and flagellar doublet microtubules: tektins, Ca2+-binding proteins, and stable protofilaments.},
journal = {J Biol Chem},
school = {the Biology Department and Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, Massachusetts 02454, and nicastro@brandeis.edu.},
year = {2014},
volume = {289},
number = {25},
pages = {17427--17444},
url = {http://dx.doi.org/10.1074/jbc.M114.568949},
doi = {10.1074/jbc.M114.568949}
}
|
| Malet H, Liu K, El Bakkouri M, Chan SWS, Effantin G, Bacia M, Houry WA and Gutsche I (2014), "Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.", Elife. Vol. 3, pp. e03653. |
| Abstract: A 3.3 MDa macromolecular cage between two Escherichia coli proteins with seemingly incompatible symmetries-the hexameric AAA+ ATPase RavA and the decameric inducible lysine decarboxylase LdcI-is reconstructed by cryo-electron microscopy to 11 Å resolution. Combined with a 7.5 Å resolution reconstruction of the minimal complex between LdcI and the LdcI-binding domain of RavA, and the previously solved crystal structures of the individual components, this work enables to build a reliable pseudoatomic model of this unusual architecture and to identify conformational rearrangements and specific elements essential for complex formation. The design of the cage created via lateral interactions between five RavA rings is unique for the diverse AAA+ ATPase superfamily. |
BibTeX:
@article{Malet2014,
author = {Malet, Hélène and Liu, Kaiyin and El Bakkouri, Majida and Chan, Sze Wah Samuel and Effantin, Gregory and Bacia, Maria and Houry, Walid A. and Gutsche, Irina},
title = {Assembly principles of a unique cage formed by hexameric and decameric E. coli proteins.},
journal = {Elife},
school = {Unit for Virus Host-Cell Interactions, Université Grenoble Alpes, Grenoble, France Unit for Virus Host-Cell Interactions, CNRS, Grenoble, France gutsche@embl.fr.},
year = {2014},
volume = {3},
pages = {e03653},
url = {http://dx.doi.org/10.7554/eLife.03653},
doi = {10.7554/eLife.03653}
}
|
| Martinez R, Schellenberger P, Vasishtan D, Aknin C, Austin S, Dacheux D, Rayne F, Siebert A, Ruzsics Z, Gruenewald K and Wodrich H (2015), "The amphipathic helix of adenovirus capsid protein VI contributes to penton release and postentry sorting.", J Virol. Vol. 89(4), pp. 2121-2135. |
| Abstract: Nuclear delivery of the adenoviral genome requires that the capsid cross the limiting membrane of the endocytic compartment and traverse the cytosol to reach the nucleus. This endosomal escape is initiated upon internalization and involves a highly coordinated process of partial disassembly of the entering capsid to release the membrane lytic internal capsid protein VI. Using wild-type and protein VI-mutated human adenovirus serotype 5 (HAdV-C5), we show that capsid stability and membrane rupture are major determinants of entry-related sorting of incoming adenovirus virions. Furthermore, by using electron cryomicroscopy, as well as penton- and protein VI-specific antibodies, we show that the amphipathic helix of protein VI contributes to capsid stability by preventing premature disassembly and deployment of pentons and protein VI. Thus, the helix has a dual function in maintaining the metastable state of the capsid by preventing premature disassembly and mediating efficient membrane lysis to evade lysosomal targeting. Based on these findings and structural data from cryo-electron microscopy, we suggest a refined disassembly mechanism upon entry.In this study, we show the intricate connection of adenovirus particle stability and the entry-dependent release of the membrane-lytic capsid protein VI required for endosomal escape. We show that the amphipathic helix of the adenovirus internal protein VI is required to stabilize pentons in the particle while coinciding with penton release upon entry and that release of protein VI mediates membrane lysis, thereby preventing lysosomal sorting. We suggest that this dual functionality of protein VI ensures an optimal disassembly process by balancing the metastable state of the mature adenovirus particle. |
BibTeX:
@article{Martinez2015,
author = {Martinez, Ruben and Schellenberger, Pascale and Vasishtan, Daven and Aknin, Cindy and Austin, Sisley and Dacheux, Denis and Rayne, Fabienne and Siebert, Alistair and Ruzsics, Zsolt and Gruenewald, Kay and Wodrich, Harald},
title = {The amphipathic helix of adenovirus capsid protein VI contributes to penton release and postentry sorting.},
journal = {J Virol},
school = {Microbiologie Fondamentale et Pathogénicité, MFP CNRS UMR 5234, Université Bordeaux, Bordeaux, France harald.wodrich@u-bordeaux2.fr.},
year = {2015},
volume = {89},
number = {4},
pages = {2121--2135},
url = {http://dx.doi.org/10.1128/JVI.02257-14},
doi = {10.1128/JVI.02257-14}
}
|
| McIntosh JR, O'Toole E, Zhudenkov K, Morphew M, Schwartz C, Ataullakhanov FI and Grishchuk EL (2013), "Conserved and divergent features of kinetochores and spindle microtubule ends from five species.", J Cell Biol. Vol. 200(4), pp. 459-474. |
| Abstract: Interfaces between spindle microtubules and kinetochores were examined in diverse species by electron tomography and image analysis. Overall structures were conserved in a mammal, an alga, a nematode, and two kinds of yeasts; all lacked dense outer plates, and most kinetochore microtubule ends flared into curved protofilaments that were connected to chromatin by slender fibrils. Analyses of curvature on >8,500 protofilaments showed that all classes of spindle microtubules displayed some flaring protofilaments, including those growing in the anaphase interzone. Curved protofilaments on anaphase kinetochore microtubules were no more flared than their metaphase counterparts, but they were longer. Flaring protofilaments in budding yeasts were linked by fibrils to densities that resembled nucleosomes; these are probably the yeast kinetochores. Analogous densities in fission yeast were larger and less well-defined, but both yeasts showed ring- or partial ring-shaped structures girding their kinetochore microtubules. Flaring protofilaments linked to chromatin are well placed to exert force on chromosomes, assuring stable attachment and reliable anaphase segregation. |
BibTeX:
@article{McIntosh2013,
author = {McIntosh, J Richard and O'Toole, Eileen and Zhudenkov, Kirill and Morphew, Mary and Schwartz, Cindi and Ataullakhanov, Fazly I. and Grishchuk, Ekaterina L.},
title = {Conserved and divergent features of kinetochores and spindle microtubule ends from five species.},
journal = {J Cell Biol},
school = {Department of Molecular, Cellular, and Developmental Biology, University of Colorado, Boulder, CO 80309, USA. richard.mcintosh@colorado.edu},
year = {2013},
volume = {200},
number = {4},
pages = {459--474},
url = {http://dx.doi.org/10.1083/jcb.201209154},
doi = {10.1083/jcb.201209154}
}
|
| Mühleip A, Kock Flygaard R, Ovciarikova J, Lacombe A, Fernandes P, Sheiner L and Amunts A (2021), "ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria.", Nature communications. Vol. 12, pp. 120. |
| Abstract: Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF . Cryo-ET and subtomogram averaging revealed that hexamers arrange into 20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa. |
BibTeX:
@article{Muehleip2021,
author = {Mühleip, Alexander and Kock Flygaard, Rasmus and Ovciarikova, Jana and Lacombe, Alice and Fernandes, Paula and Sheiner, Lilach and Amunts, Alexey},
title = {ATP synthase hexamer assemblies shape cristae of Toxoplasma mitochondria.},
journal = {Nature communications},
year = {2021},
volume = {12},
pages = {120},
doi = {10.1038/s41467-020-20381-z}
}
|
| Mühleip AW, Dewar CE, Schnaufer A, Kühlbrandt W and Davies KM (2017), "In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 114, pp. 992-997. |
| Abstract: We used electron cryotomography and subtomogram averaging to determine the in situ structures of mitochondrial ATP synthase dimers from two organisms belonging to the phylum euglenozoa: Trypanosoma brucei, a lethal human parasite, and Euglena gracilis, a photosynthetic protist. At a resolution of 32.5 Å and 27.5 Å, respectively, the two structures clearly exhibit a noncanonical F1 head, in which the catalytic (αβ)3 assembly forms a triangular pyramid rather than the pseudo-sixfold ring arrangement typical of all other ATP synthases investigated so far. Fitting of known X-ray structures reveals that this unusual geometry results from a phylum-specific cleavage of the α subunit, in which the C-terminal αC fragments are displaced by ∼20 Å and rotated by ∼30° from their expected positions. In this location, the αC fragment is unable to form the conserved catalytic interface that was thought to be essential for ATP synthesis, and cannot convert γ-subunit rotation into the conformational changes implicit in rotary catalysis. The new arrangement of catalytic subunits suggests that the mechanism of ATP generation by rotary ATPases is less strictly conserved than has been generally assumed. The ATP synthases of these organisms present a unique model system for discerning the individual contributions of the α and β subunits to the fundamental process of ATP synthesis. |
BibTeX:
@article{Muehleip2017,
author = {Mühleip, Alexander W and Dewar, Caroline E and Schnaufer, Achim and Kühlbrandt, Werner and Davies, Karen M},
title = {In situ structure of trypanosomal ATP synthase dimer reveals a unique arrangement of catalytic subunits.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2017},
volume = {114},
pages = {992--997},
doi = {10.1073/pnas.1612386114}
}
|
| Mühleip AW, Joos F, Wigge C, Frangakis AS, Kühlbrandt W and Davies KM (2016), "Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 113, pp. 8442-8447. |
| Abstract: F1Fo-ATP synthases are universal energy-converting membrane protein complexes that synthesize ATP from ADP and inorganic phosphate. In mitochondria of yeast and mammals, the ATP synthase forms V-shaped dimers, which assemble into rows along the highly curved ridges of lamellar cristae. Using electron cryotomography and subtomogram averaging, we have determined the in situ structure and organization of the mitochondrial ATP synthase dimer of the ciliate Paramecium tetraurelia. The ATP synthase forms U-shaped dimers with parallel monomers. Each complex has a prominent intracrista domain, which links the c-ring of one monomer to the peripheral stalk of the other. Close interaction of intracrista domains in adjacent dimers results in the formation of helical ATP synthase dimer arrays, which differ from the loose dimer rows in all other organisms observed so far. The parameters of the helical arrays match those of the cristae tubes, suggesting the unique features of the P. tetraurelia ATP synthase are directly responsible for generating the helical tubular cristae. We conclude that despite major structural differences between ATP synthase dimers of ciliates and other eukaryotes, the formation of ATP synthase dimer rows is a universal feature of mitochondria and a fundamental determinant of cristae morphology. |
BibTeX:
@article{Muehleip2016,
author = {Mühleip, Alexander W and Joos, Friederike and Wigge, Christoph and Frangakis, Achilleas S and Kühlbrandt, Werner and Davies, Karen M},
title = {Helical arrays of U-shaped ATP synthase dimers form tubular cristae in ciliate mitochondria.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2016},
volume = {113},
pages = {8442--8447},
doi = {10.1073/pnas.1525430113}
}
|
| Murakami K, Calero G, Brown CR, Liu X, Davis RE, Boeger H and Kornberg RD (2013), "Formation and fate of a complete 31-protein RNA polymerase II transcription preinitiation complex", Journal of Biological Chemistry. Vol. 288(9), pp. 6325-6332. ASBMB.
[BibTeX] |
BibTeX:
@article{Murakami2013a,
author = {Murakami, Kenji and Calero, Guillermo and Brown, Christopher R and Liu, Xin and Davis, Ralph E and Boeger, Hinrich and Kornberg, Roger D},
title = {Formation and fate of a complete 31-protein RNA polymerase II transcription preinitiation complex},
journal = {Journal of Biological Chemistry},
publisher = {ASBMB},
year = {2013},
volume = {288},
number = {9},
pages = {6325--6332}
}
|
| Murakami K, Elmlund H, Kalisman N, Bushnell DA, Adams CM, Azubel M, Elmlund D, Levi-Kalisman Y, Liu X, Gibbons BJ and others (2013), "Architecture of an RNA polymerase II transcription pre-initiation complex", Science. Vol. 342(6159), pp. 1238724. American Association for the Advancement of Science.
[BibTeX] |
BibTeX:
@article{Murakami2013,
author = {Murakami, Kenji and Elmlund, Hans and Kalisman, Nir and Bushnell, David A and Adams, Christopher M and Azubel, Maia and Elmlund, Dominika and Levi-Kalisman, Yael and Liu, Xin and Gibbons, Brian J and others},
title = {Architecture of an RNA polymerase II transcription pre-initiation complex},
journal = {Science},
publisher = {American Association for the Advancement of Science},
year = {2013},
volume = {342},
number = {6159},
pages = {1238724}
}
|
| Murakami K, Mattei P-J, Davis RE, Jin H, Kaplan CD and Kornberg RD (2015), "Uncoupling promoter opening from start-site scanning", Molecular cell. Vol. 59(1), pp. 133-138. Elsevier.
[BibTeX] |
BibTeX:
@article{Murakami2015,
author = {Murakami, Kenji and Mattei, Pierre-Jean and Davis, Ralph E and Jin, Huiyan and Kaplan, Craig D and Kornberg, Roger D},
title = {Uncoupling promoter opening from start-site scanning},
journal = {Molecular cell},
publisher = {Elsevier},
year = {2015},
volume = {59},
number = {1},
pages = {133--138}
}
|
| Nans A, Einheber S, Salzer JL and Stokes DL (2011), "Electron tomography of paranodal septate-like junctions and the associated axonal and glial cytoskeletons in the central nervous system.", J Neurosci Res. Vol. 89(3), pp. 310-319. |
| Abstract: The polarized domains of myelinated axons are specifically organized to maximize the efficiency of saltatory conduction. The paranodal region is directly adjacent to the node of Ranvier and contains specialized septate-like junctions that provide adhesion between axons and glial cells and that constitute a lateral diffusion barrier for nodal components. To complement and extend earlier studies on the peripheral nervous system, electron tomography was used to image paranodal regions from the central nervous system (CNS). Our three-dimensional reconstructions revealed short filamentous linkers running directly from the septate-like junctions to neurofilaments, microfilaments, and organelles within the axon. The intercellular spacing between axons and glia was measured to be 7.4 ± 0.6 nm, over twice the value previously reported in the literature (2.5-3.0 nm). Averaging of individual junctions revealed a bifurcated structure in the intercellular space that is consistent with a dimeric complex of cell adhesion molecules composing the septate-like junction. Taken together, these findings provide new insight into the structural organization of CNS paranodes and suggest that, in addition to providing axo-glial adhesion, cytoskeletal linkage to the septate-like junctions may be required to maintain axonal domains and to regulate organelle transport in myelinated axons. |
BibTeX:
@article{Nans2011,
author = {Nans, Andrea and Einheber, Steven and Salzer, James L. and Stokes, David L.},
title = {Electron tomography of paranodal septate-like junctions and the associated axonal and glial cytoskeletons in the central nervous system.},
journal = {J Neurosci Res},
school = {Skirball Institute of Biomolecular Medicine, New York University School of Medicine, New York, New York, USA.},
year = {2011},
volume = {89},
number = {3},
pages = {310--319},
url = {http://dx.doi.org/10.1002/jnr.22561},
doi = {10.1002/jnr.22561}
}
|
| Narayan T, Hayee F and Baldi A (2016), "Ai Leen Koh", Surface Engineering. Vol. 138(15), pp. 5123-5129.
[BibTeX] |
BibTeX:
@article{Narayan2016,
author = {Narayan, TC and Hayee, F and Baldi, A},
title = {Ai Leen Koh},
journal = {Surface Engineering},
year = {2016},
volume = {138},
number = {15},
pages = {5123--5129}
}
|
| Nazarov S, Bezler A, Hatzopoulos GN, Nemčíková Villímová V, Demurtas D, Le Guennec M, Guichard P and Gönczy P (2020), "Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography.", The EMBO journal. Vol. 39, pp. e106249. |
| Abstract: Centrioles are polarized microtubule-based organelles that seed the formation of cilia, and which assemble from a cartwheel containing stacked ring oligomers of SAS-6 proteins. A cryo-tomography map of centrioles from the termite flagellate Trichonympha spp. was obtained previously, but higher resolution analysis is likely to reveal novel features. Using sub-tomogram averaging (STA) in T. spp. and Trichonympha agilis, we delineate the architecture of centriolar microtubules, pinhead, and A-C linker. Moreover, we report 25 Å resolution maps of the central cartwheel, revealing notably polarized cartwheel inner densities (CID). Furthermore, STA of centrioles from the distant flagellate Teranympha mirabilis uncovers similar cartwheel architecture and a distinct filamentous CID. Fitting the CrSAS-6 crystal structure into the flagellate maps and analyzing cartwheels generated in vitro indicate that SAS-6 rings can directly stack onto one another in two alternating configurations: with a slight rotational offset and in register. Overall, improved STA maps in three flagellates enabled us to unravel novel architectural features, including of centriole polarity and cartwheel stacking, thus setting the stage for an accelerated elucidation of underlying assembly mechanisms. |
BibTeX:
@article{Nazarov2020,
author = {Nazarov, Sergey and Bezler, Alexandra and Hatzopoulos, Georgios N. and Nemčíková Villímová, Veronika and Demurtas, Davide and Le Guennec, Maeva and Guichard, Paul and Gönczy, Pierre},
title = {Novel features of centriole polarity and cartwheel stacking revealed by cryo-tomography.},
journal = {The EMBO journal},
year = {2020},
volume = {39},
pages = {e106249},
doi = {10.15252/embj.2020106249}
}
|
| Ng CT, Deng L, Chen C, Lim HH, Shi J, Surana U and Gan L (2019), "Electron cryotomography analysis of Dam1C/DASH at the kinetochore-spindle interface in situ.", The Journal of cell biology. Vol. 218, pp. 455-473. |
| Abstract: In dividing cells, depolymerizing spindle microtubules move chromosomes by pulling at their kinetochores. While kinetochore subcomplexes have been studied extensively in vitro, little is known about their in vivo structure and interactions with microtubules or their response to spindle damage. Here we combine electron cryotomography of serial cryosections with genetic and pharmacological perturbation to study the yeast chromosome segregation machinery in vivo. Each kinetochore microtubule has one (rarely, two) Dam1C/DASH outer kinetochore assemblies. Dam1C/DASH contacts the microtubule walls and does so with its flexible "bridges"; there are no contacts with the protofilaments' curved tips. In metaphase, ∼40% of the Dam1C/DASH assemblies are complete rings; the rest are partial rings. Ring completeness and binding position along the microtubule are sensitive to kinetochore attachment and tension, respectively. Our study and those of others support a model in which each kinetochore must undergo cycles of conformational change to couple microtubule depolymerization to chromosome movement. |
BibTeX:
@article{Ng2019,
author = {Ng, Cai Tong and Deng, Li and Chen, Chen and Lim, Hong Hwa and Shi, Jian and Surana, Uttam and Gan, Lu},
title = {Electron cryotomography analysis of Dam1C/DASH at the kinetochore-spindle interface in situ.},
journal = {The Journal of cell biology},
year = {2019},
volume = {218},
pages = {455--473},
doi = {10.1083/jcb.201809088}
}
|
| Ng CT and Gan L (2020), "Investigating eukaryotic cells with cryo-ET.", Molecular biology of the cell. Vol. 31, pp. 87-100. |
| Abstract: The interior of eukaryotic cells is mysterious. How do the large communities of macromolecular machines interact with each other? How do the structures and positions of these nanoscopic entities respond to new stimuli? Questions like these can now be answered with the help of a method called electron cryotomography (cryo-ET). Cryo-ET will ultimately reveal the inner workings of a cell at the protein, secondary structure, and perhaps even side-chain levels. Combined with genetic or pharmacological perturbation, cryo-ET will allow us to answer previously unimaginable questions, such as how structure, biochemistry, and forces are related in situ. Because it bridges structural biology and cell biology, cryo-ET is indispensable for structural cell biology-the study of the 3-D macromolecular structure of cells. Here we discuss some of the key ideas, strategies, auxiliary techniques, and innovations that an aspiring structural cell biologist will consider when planning to ask bold questions. |
BibTeX:
@article{Ng2020,
author = {Ng, Cai Tong and Gan, Lu},
title = {Investigating eukaryotic cells with cryo-ET.},
journal = {Molecular biology of the cell},
year = {2020},
volume = {31},
pages = {87--100},
doi = {10.1091/mbc.E18-05-0329}
}
|
| Nguyen THD, Galej WP, Bai X-c, Oubridge C, Newman AJ, Scheres SHW and Nagai K (2016), "Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution.", Nature. Vol. 530, pp. 298-302. |
| Abstract: U4/U6.U5 tri-snRNP represents a substantial part of the spliceosome before activation. A cryo-electron microscopy structure of Saccharomyces cerevisiae U4/U6.U5 tri-snRNP at 3.7 Å resolution led to an essentially complete atomic model comprising 30 proteins plus U4/U6 and U5 small nuclear RNAs (snRNAs). The structure reveals striking interweaving interactions of the protein and RNA components, including extended polypeptides penetrating into subunit interfaces. The invariant ACAGAGA sequence of U6 snRNA, which base-pairs with the 5'-splice site during catalytic activation, forms a hairpin stabilized by Dib1 and Prp8 while the adjacent nucleotides interact with the exon binding loop 1 of U5 snRNA. Snu114 harbours GTP, but its putative catalytic histidine is held away from the γ-phosphate by hydrogen bonding to a tyrosine in the amino-terminal domain of Prp8. Mutation of this histidine to alanine has no detectable effect on yeast growth. The structure provides important new insights into the spliceosome activation process leading to the formation of the catalytic centre. |
BibTeX:
@article{Nguyen2016,
author = {Nguyen, Thi Hoang Duong and Galej, Wojciech P and Bai, Xiao-chen and Oubridge, Chris and Newman, Andrew J and Scheres, Sjors H W and Nagai, Kiyoshi},
title = {Cryo-EM structure of the yeast U4/U6.U5 tri-snRNP at 3.7 Å resolution.},
journal = {Nature},
year = {2016},
volume = {530},
pages = {298--302},
doi = {10.1038/nature16940}
}
|
| Ni T, Frosio T, Mendonça L, Sheng Y, Clare D, Himes BA and Zhang P (2022), "High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.", Nature protocols. |
| Abstract: Cryo-electron tomography and subtomogram averaging (STA) has developed rapidly in recent years. It provides structures of macromolecular complexes in situ and in cellular context at or below subnanometer resolution and has led to unprecedented insights into the inner working of molecular machines in their native environment, as well as their functional relevant conformations and spatial distribution within biological cells or tissues. Given the tremendous potential of cryo-electron tomography STA in in situ structural cell biology, we previously developed emClarity, a graphics processing unit-accelerated image-processing software that offers STA and classification of macromolecular complexes at high resolution. However, the workflow remains challenging, especially for newcomers to the field. In this protocol, we describe a detailed workflow, processing and parameters associated with each step, from initial tomography tilt-series data to the final 3D density map, with several features unique to emClarity. We use four different samples, including human immunodeficiency virus type 1 Gag assemblies, ribosome and apoferritin, to illustrate the procedure and results of STA and classification. Following the processing steps described in this protocol, along with a comprehensive tutorial and guidelines for troubleshooting and parameter optimization, one can obtain density maps up to 2.8 Å resolution from six tilt series by cryo-electron tomography STA. |
BibTeX:
@article{Ni2022,
author = {Ni, Tao and Frosio, Thomas and Mendonça, Luiza and Sheng, Yuewen and Clare, Daniel and Himes, Benjamin A. and Zhang, Peijun},
title = {High-resolution in situ structure determination by cryo-electron tomography and subtomogram averaging using emClarity.},
journal = {Nature protocols},
year = {2022},
doi = {10.1038/s41596-021-00648-5}
}
|
| Nicastro D, Fu X, Heuser T, Tso A, Porter ME and Linck RW (2011), "Cryo-electron tomography reveals conserved features of doublet microtubules in flagella.", Proc Natl Acad Sci U S A. Vol. 108(42), pp. E845-E853. |
| Abstract: The axoneme forms the essential and conserved core of cilia and flagella. We have used cryo-electron tomography of Chlamydomonas and sea urchin flagella to answer long-standing questions and to provide information about the structure of axonemal doublet microtubules (DMTs). Solving an ongoing controversy, we show that B-tubules of DMTs contain exactly 10 protofilaments (PFs) and that the inner junction (IJ) and outer junction between the A- and B-tubules are fundamentally different. The outer junction, crucial for the initiation of doublet formation, appears to be formed by close interactions between the tubulin subunits of three PFs with unusual tubulin interfaces; other investigators have reported that this junction is weakened by mutations affecting posttranslational modifications of tubulin. The IJ consists of an axially periodic ladder-like structure connecting tubulin PFs of the A- and B-tubules. The recently discovered microtubule inner proteins (MIPs) on the inside of the A- and B-tubules are more complex than previously thought. They are composed of alternating small and large subunits with periodicities of 16 and/or 48 nm. MIP3 forms arches connecting B-tubule PFs, contrary to an earlier report that MIP3 forms the IJ. Finally, the "beak" structures within the B-tubules of Chlamydomonas DMT1, DMT5, and DMT6 are clearly composed of a longitudinal band of proteins repeating with a periodicity of 16 nm. These findings, discussed in relation to genetic and biochemical data, provide a critical foundation for future work on the molecular assembly and stability of the axoneme, as well as its function in motility and sensory transduction. |
BibTeX:
@article{Nicastro2011,
author = {Nicastro, Daniela and Fu, Xiaofeng and Heuser, Thomas and Tso, Alan and Porter, Mary E. and Linck, Richard W.},
title = {Cryo-electron tomography reveals conserved features of doublet microtubules in flagella.},
journal = {Proc Natl Acad Sci U S A},
school = {Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02454, USA. nicastro@brandeis.edu},
year = {2011},
volume = {108},
number = {42},
pages = {E845--E853},
url = {http://dx.doi.org/10.1073/pnas.1106178108},
doi = {10.1073/pnas.1106178108}
}
|
| Nicastro D, Schwartz C, Pierson J, Gaudette R, Porter ME and McIntosh JR (2006), "The molecular architecture of axonemes revealed by cryoelectron tomography.", Science. Vol. 313(5789), pp. 944-948. |
| Abstract: Eukaryotic flagella and cilia are built on a 9 + 2 array of microtubules plus >250 accessory proteins, forming a biological machine called the axoneme. Here we describe the three-dimensional structure of rapidly frozen axonemes from Chlamydomonas and sea urchin sperm, using cryoelectron tomography and image processing to focus on the motor enzyme dynein. Our images suggest a model for the way dynein generates force to slide microtubules. They also reveal two dynein linkers that may provide "hard-wiring" to coordinate motor enzyme action, both circumferentially and along the axoneme. Periodic densities were also observed inside doublet microtubules; these may contribute to doublet stability. |
BibTeX:
@article{Nicastro2006,
author = {Nicastro, Daniela and Schwartz, Cindi and Pierson, Jason and Gaudette, Richard and Porter, Mary E. and McIntosh, J Richard},
title = {The molecular architecture of axonemes revealed by cryoelectron tomography.},
journal = {Science},
school = {Laboratory for 3D Electron Microscopy of Cells, Department of Molecular, Cellular, and Developmental Biology, CB 347, University of Colorado, Boulder, CO 80309-0347, USA. nicastro@colorado.edu},
year = {2006},
volume = {313},
number = {5789},
pages = {944--948},
url = {http://dx.doi.org/10.1126/science.1128618},
doi = {10.1126/science.1128618}
}
|
| Obr M and Schur FKM (2019), "Structural analysis of pleomorphic and asymmetric viruses using cryo-electron tomography and subtomogram averaging.", Advances in virus research. Vol. 105, pp. 117-159. |
| Abstract: Describing the protein interactions that form pleomorphic and asymmetric viruses represents a considerable challenge to most structural biology techniques, including X-ray crystallography and single particle cryo-electron microscopy. Obtaining a detailed understanding of these interactions is nevertheless important, considering the number of relevant human pathogens that do not follow strict icosahedral or helical symmetry. Cryo-electron tomography and subtomogram averaging methods provide structural insights into complex biological environments and are well suited to go beyond structures of perfectly symmetric viruses. This chapter discusses recent developments showing that cryo-ET and subtomogram averaging can provide high-resolution insights into hitherto unknown structural features of pleomorphic and asymmetric virus particles. It also describes how these methods have significantly added to our understanding of retrovirus capsid assemblies in immature and mature viruses. Additional examples of irregular viruses and their associated proteins, whose structures have been studied via cryo-ET and subtomogram averaging, further support the versatility of these methods. |
BibTeX:
@article{Obr2019,
author = {Obr, Martin and Schur, Florian K M},
title = {Structural analysis of pleomorphic and asymmetric viruses using cryo-electron tomography and subtomogram averaging.},
journal = {Advances in virus research},
year = {2019},
volume = {105},
pages = {117--159},
doi = {10.1016/bs.aivir.2019.07.008}
}
|
| Oda T, Abe T, Yanagisawa H and Kikkawa M (2016), "Docking-complex-independent alignment of Chlamydomonas outer dynein arms with 24-nm periodicity in vitro.", J Cell Sci. Vol. 129(8), pp. 1547-1551. |
| Abstract: The docking complex is a molecular complex necessary for assembly of outer dynein arms (ODAs) on the axonemal doublet microtubules (DMTs) in cilia and flagella. The docking complex is hypothesized to be a 24-nm molecular ruler because ODAs align along the DMTs with 24-nm periodicity. In this study, we rigorously tested this hypothesis using structural and genetic methods. We found that the ODAs can bind to DMTs and porcine microtubules with 24-nm periodicities even in the absence of the docking complexin vitro Using cryo-electron tomography and structural labeling, we observed that the docking complex took an unexpectedly flexible conformation and did not lie along the length of DMTs. In the absence of docking complex, ODAs were released from the DMT at relatively low ionic strength conditions, suggesting that the docking complex strengthens the electrostatic interactions between the ODA and DMT. Based on these results, we conclude that the docking complex serves as a flexible stabilizer of the ODA rather than as a molecular ruler. |
BibTeX:
@article{Oda2016,
author = {Oda, Toshiyuki and Abe, Tatsuki and Yanagisawa, Haruaki and Kikkawa, Masahide},
title = {Docking-complex-independent alignment of Chlamydomonas outer dynein arms with 24-nm periodicity in vitro.},
journal = {J Cell Sci},
school = {Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.},
year = {2016},
volume = {129},
number = {8},
pages = {1547--1551},
url = {http://dx.doi.org/10.1242/jcs.184598},
doi = {10.1242/jcs.184598}
}
|
| Oda T, Abe T, Yanagisawa H and Kikkawa M (2016), "Structure and function of outer dynein arm intermediate and light chain complex.", Mol Biol Cell. Vol. 27(7), pp. 1051-1059. |
| Abstract: The outer dynein arm (ODA) is a molecular complex that drives the beating motion of cilia/flagella.ChlamydomonasODA is composed of three heavy chains (HCs), two ICs, and 11 light chains (LCs). Although the three-dimensional (3D) structure of the whole ODA complex has been investigated, the 3D configurations of the ICs and LCs are largely unknown. Here we identified the 3D positions of the two ICs and three LCs using cryo-electron tomography and structural labeling. We found that these ICs and LCs were all localized at the root of the outer-inner dynein (OID) linker, designated the ODA-Beak complex. Of interest, the coiled-coil domain of IC2 extended from the ODA-Beak to the outer surface of ODA. Furthermore, we investigated the molecular mechanisms of how the OID linker transmits signals to the ODA-Beak, by manipulating the interaction within the OID linker using a chemically induced dimerization system. We showed that the cross-linking of the OID linker strongly suppresses flagellar motility in vivo. These results suggest that the ICs and LCs of the ODA form the ODA-Beak, which may be involved in mechanosignaling from the OID linker to the HCs. |
BibTeX:
@article{Oda2016b,
author = {Oda, Toshiyuki and Abe, Tatsuki and Yanagisawa, Haruaki and Kikkawa, Masahide},
title = {Structure and function of outer dynein arm intermediate and light chain complex.},
journal = {Mol Biol Cell},
school = {Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, Tokyo 113-0033, Japan.},
year = {2016},
volume = {27},
number = {7},
pages = {1051--1059},
url = {http://dx.doi.org/10.1091/mbc.E15-10-0723},
doi = {10.1091/mbc.E15-10-0723}
}
|
| Oda T and Kikkawa M (2013), "Novel structural labeling method using cryo-electron tomography and biotin-streptavidin system.", J Struct Biol. Vol. 183(3), pp. 305-311. |
| Abstract: There are a number of large macromolecular complexes that play important roles in the cell, and identifying the positions of their components is a key step to understanding their structure and function. Several structural labeling methods have been applied to electron microscopy in order to locate a specific component within a macromolecular complex, but each method is associated with problems in specificity, occupancy, signal intensity or precision. Here, we report a novel method for identifying the 3D locations of proteins using biotin-streptavidin labeling and cryo-electron tomography. We labeled a biotinylation-tagged intermediate chain of an axonemal dynein by streptavidin within the Chlamydomonas axoneme and visualized the 3D positions of the labels using subtomogram averaging. Increase of the density attributed to the bound streptavidin was validated by Student's t-test. In conclusion, the combination of the biotin-streptavidin system and cryo-electron tomography is a powerful method to investigate the structure of large macromolecular complexes. |
BibTeX:
@article{Oda2013,
author = {Oda, Toshiyuki and Kikkawa, Masahide},
title = {Novel structural labeling method using cryo-electron tomography and biotin-streptavidin system.},
journal = {J Struct Biol},
school = {Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.},
year = {2013},
volume = {183},
number = {3},
pages = {305--311},
url = {http://dx.doi.org/10.1016/j.jsb.2013.07.003},
doi = {10.1016/j.jsb.2013.07.003}
}
|
| Oda T and Yanagisawa H (2020), "Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs.", Communications biology. Vol. 3, pp. 585. |
| Abstract: The Z-disc forms a boundary between sarcomeres, which constitute structural and functional units of striated muscle tissue. Actin filaments from adjacent sarcomeres are cross-bridged by α-actinin in the Z-disc, allowing transmission of tension across the myofibril. Despite decades of studies, the 3D structure of Z-disc has remained elusive due to the limited resolution of conventional electron microscopy. Here, we observed porcine cardiac myofibrils using cryo-electron tomography and reconstructed the 3D structures of the actin-actinin cross-bridging complexes within the Z-discs in relaxed and activated states. We found that the α-actinin dimers showed contraction-dependent swinging and sliding motions in response to a global twist in the F-actin lattice. Our observation suggests that the actin-actinin complex constitutes a molecular lattice spring, which maintains the integrity of the Z-disc during the muscle contraction cycle. |
BibTeX:
@article{Oda2020,
author = {Oda, Toshiyuki and Yanagisawa, Haruaki},
title = {Cryo-electron tomography of cardiac myofibrils reveals a 3D lattice spring within the Z-discs.},
journal = {Communications biology},
year = {2020},
volume = {3},
pages = {585},
doi = {10.1038/s42003-020-01321-5}
}
|
| Oda T, Yanagisawa H and Kikkawa M (2015), "Detailed structural and biochemical characterization of the nexin-dynein regulatory complex.", Mol Biol Cell. Vol. 26(2), pp. 294-304. |
| Abstract: The nexin-dynein regulatory complex (N-DRC) forms a cross-bridge between the outer doublet microtubules of the axoneme and regulates dynein motor activity in cilia/flagella. Although the molecular composition and the three-dimensional structure of N-DRC have been studied using mutant strains lacking N-DRC subunits, more accurate approaches are necessary to characterize the structure and function of N-DRC. In this study, we precisely localized DRC1, DRC2, and DRC4 using cryo-electron tomography and structural labeling. All three N-DRC subunits had elongated conformations and spanned the length of N-DRC. Furthermore, we purified N-DRC and characterized its microtubule-binding properties. Purified N-DRC bound to the microtubule and partially inhibited microtubule sliding driven by the outer dynein arms (ODAs). Of interest, microtubule sliding was observed even in the presence of fourfold molar excess of N-DRC relative to ODA. These results provide insights into the role of N-DRC in generating the beating motions of cilia/flagella. |
BibTeX:
@article{Oda2015,
author = {Oda, Toshiyuki and Yanagisawa, Haruaki and Kikkawa, Masahide},
title = {Detailed structural and biochemical characterization of the nexin-dynein regulatory complex.},
journal = {Mol Biol Cell},
school = {Department of Cell Biology and Anatomy, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo Bunkyo-ku, Tokyo 113-0033, Japan.},
year = {2015},
volume = {26},
number = {2},
pages = {294--304},
url = {http://dx.doi.org/10.1091/mbc.E14-09-1367},
doi = {10.1091/mbc.E14-09-1367}
}
|
| Oda T, Yanagisawa H, Shinmori H, Ogawa Y and Kawamura T (2022), "Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation", eLife. Vol. 11 eLife Sciences Publications Ltd. |
| Abstract: Langerhans cells are specialized antigen-presenting cells localized within the epidermis and mucosal epithelium. Upon contact with Langerhans cells, pathogens are captured by the C-type lectin langerin and internalized into a structurally unique vesicle known as a Birbeck granule. Although the immunological role of Langerhans cells and Birbeck granules have been extensively studied, the mechanism by which the characteristic zippered membrane structure of Birbeck granules is formed remains elusive. In this study, we observed isolated Birbeck granules using cryo-electron tomography and reconstructed the 3D structure of the repeating unit of the honeycomb lattice of langerin at 6.4 Å resolution. We found that the interaction between the two langerin trimers was mediated by docking the flexible loop at residues 258–263 into the secondary carbohydrate-binding cleft. Mutations within the loop inhibited Birbeck granule formation and the internalization of HIV pseudovirus. These findings suggest a molecular mechanism for membrane zippering during Birbeck granule biogenesis and provide insight into the role of langerin in the defense against viral infection. |
BibTeX:
@article{Oda2022,
author = {Toshiyuki Oda and Haruaki Yanagisawa and Hideyuki Shinmori and Youichi Ogawa and Tatsuyoshi Kawamura},
title = {Cryo-electron tomography of Birbeck granules reveals the molecular mechanism of langerin lattice formation},
journal = {eLife},
publisher = {eLife Sciences Publications Ltd},
year = {2022},
volume = {11},
url = {https://elifesciences.org/articles/79990},
doi = {10.7554/eLife.79990}
}
|
| Oda T, Yanagisawa H, Yagi T and Kikkawa M (2014), "Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity.", J Cell Biol. Vol. 204(5), pp. 807-819. |
| Abstract: Cilia/flagella are conserved organelles that generate fluid flow in eukaryotes. The bending motion of flagella requires concerted activity of dynein motors. Although it has been reported that the central pair apparatus (CP) and radial spokes (RSs) are important for flagellar motility, the molecular mechanism underlying CP- and RS-mediated dynein regulation has not been identified. In this paper, we identified nonspecific intermolecular collision between CP and RS as one of the regulatory mechanisms for flagellar motility. By combining cryoelectron tomography and motility analyses of Chlamydomonas reinhardtii flagella, we show that binding of streptavidin to RS heads paralyzed flagella. Moreover, the motility defect in a CP projection mutant could be rescued by the addition of exogenous protein tags on RS heads. Genetic experiments demonstrated that outer dynein arms are the major downstream effectors of CP- and RS-mediated regulation of flagellar motility. These results suggest that mechanosignaling between CP and RS regulates dynein activity in eukaryotic flagella. |
BibTeX:
@article{Oda2014,
author = {Oda, Toshiyuki and Yanagisawa, Haruaki and Yagi, Toshiki and Kikkawa, Masahide},
title = {Mechanosignaling between central apparatus and radial spokes controls axonemal dynein activity.},
journal = {J Cell Biol},
school = {Department of Cell Biology and Anatomy, Graduate School of Medicine, The University of Tokyo, Bunkyo-ku, Tokyo 113-0033, Japan.},
year = {2014},
volume = {204},
number = {5},
pages = {807--819},
url = {http://dx.doi.org/10.1083/jcb.201312014},
doi = {10.1083/jcb.201312014}
}
|
| Olcese C, Patel MP, Shoemark A, Kiviluoto S, Legendre M, Williams HJ, Vaughan CK, Hayward J, Goldenberg A, Emes RD, Munye MM, Dyer L, Cahill T, Bevillard J, Gehrig C, Guipponi M, Chantot S, Duquesnoy P, Thomas L, Jeanson L, Copin B, Tamalet A, Thauvin-Robinet C, Papon J-F, Garin A, Pin I, Vera G, Aurora P, Fassad MR, Jenkins L, Boustred C, Cullup T, Dixon M, Onoufriadis A, Bush A, Chung EMK, Antonarakis SE, Loebinger MR, Wilson R, Armengot M, Escudier E, Hogg C, Group UR, Amselem S, Sun Z, Bartoloni L, Blouin J-L and Mitchison HM (2017), "X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.", Nature communications. Vol. 8, pp. 14279. |
| Abstract: By moving essential body fluids and molecules, motile cilia and flagella govern respiratory mucociliary clearance, laterality determination and the transport of gametes and cerebrospinal fluid. Primary ciliary dyskinesia (PCD) is an autosomal recessive disorder frequently caused by non-assembly of dynein arm motors into cilia and flagella axonemes. Before their import into cilia and flagella, multi-subunit axonemal dynein arms are thought to be stabilized and pre-assembled in the cytoplasm through a DNAAF2-DNAAF4-HSP90 complex akin to the HSP90 co-chaperone R2TP complex. Here, we demonstrate that large genomic deletions as well as point mutations involving PIH1D3 are responsible for an X-linked form of PCD causing disruption of early axonemal dynein assembly. We propose that PIH1D3, a protein that emerges as a new player of the cytoplasmic pre-assembly pathway, is part of a complementary conserved R2TP-like HSP90 co-chaperone complex, the loss of which affects assembly of a subset of inner arm dyneins. |
BibTeX:
@article{Olcese2017,
author = {Olcese, Chiara and Patel, Mitali P and Shoemark, Amelia and Kiviluoto, Santeri and Legendre, Marie and Williams, Hywel J and Vaughan, Cara K and Hayward, Jane and Goldenberg, Alice and Emes, Richard D and Munye, Mustafa M and Dyer, Laura and Cahill, Thomas and Bevillard, Jeremy and Gehrig, Corinne and Guipponi, Michel and Chantot, Sandra and Duquesnoy, Philippe and Thomas, Lucie and Jeanson, Ludovic and Copin, Bruno and Tamalet, Aline and Thauvin-Robinet, Christel and Papon, Jean-François and Garin, Antoine and Pin, Isabelle and Vera, Gabriella and Aurora, Paul and Fassad, Mahmoud R and Jenkins, Lucy and Boustred, Christopher and Cullup, Thomas and Dixon, Mellisa and Onoufriadis, Alexandros and Bush, Andrew and Chung, Eddie M K and Antonarakis, Stylianos E and Loebinger, Michael R and Wilson, Robert and Armengot, Miguel and Escudier, Estelle and Hogg, Claire and UK10K Rare Group and Amselem, Serge and Sun, Zhaoxia and Bartoloni, Lucia and Blouin, Jean-Louis and Mitchison, Hannah M},
title = {X-linked primary ciliary dyskinesia due to mutations in the cytoplasmic axonemal dynein assembly factor PIH1D3.},
journal = {Nature communications},
year = {2017},
volume = {8},
pages = {14279},
doi = {10.1038/ncomms14279}
}
|
| Owa M, Uchihashi T, Yanagisawa H-A, Yamano T, Iguchi H, Fukuzawa H, Wakabayashi K-I, Ando T and Kikkawa M (2019), "Inner lumen proteins stabilize doublet microtubules in cilia and flagella.", Nature communications. Vol. 10, pp. 1143. |
| Abstract: Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes. |
BibTeX:
@article{Owa2019,
author = {Owa, Mikito and Uchihashi, Takayuki and Yanagisawa, Haru-Aki and Yamano, Takashi and Iguchi, Hiro and Fukuzawa, Hideya and Wakabayashi, Ken-Ichi and Ando, Toshio and Kikkawa, Masahide},
title = {Inner lumen proteins stabilize doublet microtubules in cilia and flagella.},
journal = {Nature communications},
year = {2019},
volume = {10},
pages = {1143},
doi = {10.1038/s41467-019-09051-x}
}
|
| van Pee K, Neuhaus A, D'Imprima E, Mills DJ, Kühlbrandt W and Yildiz Ö (2017), "CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin.", eLife. Vol. 6 |
| Abstract: Many pathogenic bacteria produce pore-forming toxins to attack and kill human cells. We have determined the 4.5 Å structure of the 2.2 MDa pore complex of pneumolysin, the main virulence factor of Streptococcus pneumoniae, by cryoEM. The pneumolysin pore is a 400 Å ring of 42 membrane-inserted monomers. Domain 3 of the soluble toxin refolds into two 85 Å β-hairpins that traverse the lipid bilayer and assemble into a 168-strand β-barrel. The pore complex is stabilized by salt bridges between β-hairpins of adjacent subunits and an internal α-barrel. The apolar outer barrel surface with large sidechains is immersed in the lipid bilayer, while the inner barrel surface is highly charged. Comparison of the cryoEM pore complex to the prepore structure obtained by electron cryo-tomography and the x-ray structure of the soluble form reveals the detailed mechanisms by which the toxin monomers insert into the lipid bilayer to perforate the target membrane. |
BibTeX:
@article{Pee2017,
author = {van Pee, Katharina and Neuhaus, Alexander and D'Imprima, Edoardo and Mills, Deryck J and Kühlbrandt, Werner and Yildiz, Özkan},
title = {CryoEM structures of membrane pore and prepore complex reveal cytolytic mechanism of Pneumolysin.},
journal = {eLife},
year = {2017},
volume = {6},
doi = {10.7554/eLife.23644}
}
|
| Pilhofer M, Ladinsky MS, McDowall AW, Petroni G and Jensen GJ (2011), "Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton.", PLoS Biol. Vol. 9(12), pp. e1001213. |
| Abstract: Microtubules play crucial roles in cytokinesis, transport, and motility, and are therefore superb targets for anti-cancer drugs. All tubulins evolved from a common ancestor they share with the distantly related bacterial cell division protein FtsZ, but while eukaryotic tubulins evolved into highly conserved microtubule-forming heterodimers, bacterial FtsZ presumably continued to function as single homopolymeric protofilaments as it does today. Microtubules have not previously been found in bacteria, and we lack insight into their evolution from the tubulin/FtsZ ancestor. Using electron cryomicroscopy, here we show that the tubulin homologs BtubA and BtubB form microtubules in bacteria and suggest these be referred to as "bacterial microtubules" (bMTs). bMTs share important features with their eukaryotic counterparts, such as straight protofilaments and similar protofilament interactions. bMTs are composed of only five protofilaments, however, instead of the 13 typical in eukaryotes. These and other results suggest that rather than being derived from modern eukaryotic tubulin, BtubA and BtubB arose from early tubulin intermediates that formed small microtubules. Since we show that bacterial microtubules can be produced in abundance in vitro without chaperones, they should be useful tools for tubulin research and drug screening. |
BibTeX:
@article{Pilhofer2011,
author = {Pilhofer, Martin and Ladinsky, Mark S. and McDowall, Alasdair W. and Petroni, Giulio and Jensen, Grant J.},
title = {Microtubules in bacteria: Ancient tubulins build a five-protofilament homolog of the eukaryotic cytoskeleton.},
journal = {PLoS Biol},
school = {California Institute of Technology, Pasadena, California, USA. martin-pilhofer@web.de},
year = {2011},
volume = {9},
number = {12},
pages = {e1001213},
url = {http://dx.doi.org/10.1371/journal.pbio.1001213},
doi = {10.1371/journal.pbio.1001213}
}
|
| Pinskey JM, Lagisetty A, Gui L, Phan N, Reetz E, Tavakoli A, Fu G and Nicastro D (2022), "Three-dimensional flagella structures from animals' closest unicellular relatives, the Choanoflagellates.", eLife. Vol. 11 |
| Abstract: In most eukaryotic organisms, cilia and flagella perform a variety of life-sustaining roles related to environmental sensing and motility. Cryo-electron microscopy has provided considerable insight into the morphology and function of flagellar structures, but studies have been limited to less than a dozen of the millions of known eukaryotic species. Ultrastructural information is particularly lacking for unicellular organisms in the Opisthokonta clade, leaving a sizeable gap in our understanding of flagella evolution between unicellular species and multicellular metazoans (animals). Choanoflagellates are important aquatic heterotrophs, uniquely positioned within the opisthokonts as the metazoans' closest living unicellular relatives. We performed cryo-focused ion beam milling and cryo-electron tomography on flagella from the choanoflagellate species . We show that the axonemal dyneins, radial spokes, and central pair complex in more closely resemble metazoan structures than those of unicellular organisms from other suprakingdoms. In addition, we describe unique features of flagella, including microtubule holes, microtubule inner proteins, and the flagellar vane: a fine, net-like extension that has been notoriously difficult to visualize using other methods. Furthermore, we report barb-like structures of unknown function on the extracellular surface of the flagellar membrane. Together, our findings provide new insights into choanoflagellate biology and flagella evolution between unicellular and multicellular opisthokonts. |
BibTeX:
@article{Pinskey2022,
author = {Pinskey, Justine M. and Lagisetty, Adhya and Gui, Long and Phan, Nhan and Reetz, Evan and Tavakoli, Amirrasoul and Fu, Gang and Nicastro, Daniela},
title = {Three-dimensional flagella structures from animals' closest unicellular relatives, the Choanoflagellates.},
journal = {eLife},
year = {2022},
volume = {11},
doi = {10.7554/eLife.78133}
}
|
| Prasad VM, Leaman DP, Lovendahl KN, Croft JT, Benhaim MA, Hodge EA, Zwick MB and Lee KK (), "Cryo-ET of HIV reveals Env positioning on Gag lattice and structural variation among Env trimers". |
BibTeX:
@misc{Prasad,
author = {Vidya Mangala Prasad and Daniel P. Leaman and Klaus N. Lovendahl and Jacob T. Croft and Mark A. Benhaim and Edgar A. Hodge and Michael B. Zwick and Kelly K. Lee},
title = {Cryo-ET of HIV reveals Env positioning on Gag lattice and structural variation among Env trimers},
doi = {10.1101/2021.08.31.458345}
}
|
| Punch EK, Hover S, Blest HTW, Fuller J, Hewson R, Fontana J, Mankouri J and Barr JN (2018), "Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus.", The Journal of biological chemistry. Vol. 293, pp. 9937-9944. |
| Abstract: Many enveloped viruses enter cells through the endocytic network, from which they must subsequently escape through fusion of viral and endosomal membranes. This membrane fusion is mediated by virus-encoded spikes that respond to the dynamic endosomal environment, which triggers conformational changes in the spikes that initiate the fusion process. Several fusion triggers have been identified and include pH, membrane composition, and endosome-resident proteins, and these cues dictate when and where viral fusion occurs. We recently reported that infection with an enveloped bunyavirus requires elevated potassium ion concentrations [K ], controlled by cellular K channels, that are encountered during viral transit through maturing endosomes. Here we reveal the molecular basis for the K requirement of bunyaviruses through the first direct visualization of a member of the Nairoviridae family, namely Hazara virus (HAZV), using cryo-EM. Using cryo-electron tomography, we observed HAZV spike glycoproteins within infectious HAZV particles exposed to both high and low [K ], which showed that exposure to K alone results in dramatic changes to the ultrastructural architecture of the virion surface. In low [K ], the spikes adopted a compact conformation arranged in locally ordered arrays, whereas, following exposure to high [K ], the spikes became extended, and spike-membrane interactions were observed. Viruses exposed to high [K ] also displayed enhanced infectivity, thus identifying K as a newly defined trigger that helps promote viral infection. Finally, we confirmed that K channel blockers are inhibitory to HAZV infection, highlighting the potential of K channels as anti-bunyavirus targets. |
BibTeX:
@article{Punch2018,
author = {Punch, Emma K and Hover, Samantha and Blest, Henry T W and Fuller, Jack and Hewson, Roger and Fontana, Juan and Mankouri, Jamel and Barr, John N},
title = {Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus.},
journal = {The Journal of biological chemistry},
year = {2018},
volume = {293},
pages = {9937--9944},
doi = {10.1074/jbc.RA118.002494}
}
|
| Punch EK, Hover S, Blest HTW, Fuller J, Hewson R, Fontana J, Mankouri J and Barr JN (2019), "Correction: Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus.", The Journal of biological chemistry. Vol. 294, pp. 2579. |
BibTeX:
@article{Punch2019,
author = {Punch, Emma K and Hover, Samantha and Blest, Henry T W and Fuller, Jack and Hewson, Roger and Fontana, Juan and Mankouri, Jamel and Barr, John N},
title = {Correction: Potassium is a trigger for conformational change in the fusion spike of an enveloped RNA virus.},
journal = {The Journal of biological chemistry},
year = {2019},
volume = {294},
pages = {2579},
doi = {10.1074/jbc.AAC119.007718}
}
|
| Pyle E and Zanetti G (2021), "Current data processing strategies for cryo-electron tomography and subtomogram averaging.", The Biochemical journal. Vol. 478, pp. 1827-1845. |
| Abstract: Cryo-electron tomography (cryo-ET) can be used to reconstruct three-dimensional (3D) volumes, or tomograms, from a series of tilted two-dimensional images of biological objects in their near-native states in situ or in vitro. 3D subvolumes, or subtomograms, containing particles of interest can be extracted from tomograms, aligned, and averaged in a process called subtomogram averaging (STA). STA overcomes the low signal to noise ratio within the individual subtomograms to generate structures of the particle(s) of interest. In recent years, cryo-ET with STA has increasingly been capable of reaching subnanometer resolution due to improvements in microscope hardware and data processing strategies. There has also been an increase in the number and quality of software packages available to process cryo-ET data with STA. In this review, we describe and assess the data processing strategies available for cryo-ET data and highlight the recent software developments which have enabled the extraction of high-resolution information from cryo-ET datasets. |
BibTeX:
@article{Pyle2021,
author = {Pyle, Euan and Zanetti, Giulia},
title = {Current data processing strategies for cryo-electron tomography and subtomogram averaging.},
journal = {The Biochemical journal},
year = {2021},
volume = {478},
pages = {1827--1845},
doi = {10.1042/BCJ20200715}
}
|
| Rao Q, Han L, Wang Y, Chai P, Kuo Y-W, Yang R, Hu F, Yang Y, Howard J and Zhang K (2021), "Structures of outer-arm dynein array on microtubule doublet reveal a motor coordination mechanism.", Nature structural & molecular biology. Vol. 28, pp. 799-810. |
| Abstract: Thousands of outer-arm dyneins (OADs) are arrayed in the axoneme to drive a rhythmic ciliary beat. Coordination among multiple OADs is essential for generating mechanical forces to bend microtubule doublets (MTDs). Using electron microscopy, we determined high-resolution structures of Tetrahymena thermophila OAD arrays bound to MTDs in two different states. OAD preferentially binds to MTD protofilaments with a pattern resembling the native tracks for its distinct microtubule-binding domains. Upon MTD binding, free OADs are induced to adopt a stable parallel conformation, primed for array formation. Extensive tail-to-head (TTH) interactions between OADs are observed, which need to be broken for ATP turnover by the dynein motor. We propose that OADs in an array sequentially hydrolyze ATP to slide the MTDs. ATP hydrolysis in turn relaxes the TTH interfaces to effect free nucleotide cycles of downstream OADs. These findings lead to a model explaining how conformational changes in the axoneme produce coordinated action of dyneins. |
BibTeX:
@article{Rao2021,
author = {Rao, Qinhui and Han, Long and Wang, Yue and Chai, Pengxin and Kuo, Yin-Wei and Yang, Renbin and Hu, Fangheng and Yang, Yuchen and Howard, Jonathon and Zhang, Kai},
title = {Structures of outer-arm dynein array on microtubule doublet reveal a motor coordination mechanism.},
journal = {Nature structural & molecular biology},
year = {2021},
volume = {28},
pages = {799--810},
doi = {10.1038/s41594-021-00656-9}
}
|
| Rapisarda C, Cherrak Y, Kooger R, Schmidt V, Pellarin R, Logger L, Cascales E, Pilhofer M, Durand E and Fronzes R (2019), "javax.xml.bind.JAXBElement@33b05f3, and high-resolution cryo-EM structure of a bacterial type VI secretion system membrane complex.", The EMBO journal. Vol. 38 |
| Abstract: Bacteria have evolved macromolecular machineries that secrete effectors and toxins to survive and thrive in diverse environments. The type VI secretion system (T6SS) is a contractile machine that is related to phages. It is composed of a phage tail-like structure inserted in the bacterial cell envelope by a membrane complex (MC) comprising the TssJ, TssL and TssM proteins. We previously reported the low-resolution negative-stain electron microscopy structure of the enteroaggregative MC and proposed a rotational 5-fold symmetry with a TssJ:TssL:TssM stoichiometry of 2:2:2. Here, cryo-electron tomography analyses of the T6SS MC confirm the 5-fold symmetry and identify the regions of the structure that insert into the bacterial membranes. A high-resolution model obtained by single-particle cryo-electron microscopy highlights new features: five additional copies of TssJ, yielding a TssJ:TssL:TssM stoichiometry of 3:2:2, an 11-residue loop in TssM, protruding inside the lumen of the MC and constituting a functionally important periplasmic gate, and hinge regions. Based on these data, we propose an updated model on MC structure and dynamics during T6SS assembly and function. |
BibTeX:
@article{Rapisarda2019,
author = {Rapisarda, Chiara and Cherrak, Yassine and Kooger, Romain and Schmidt, Victoria and Pellarin, Riccardo and Logger, Laureen and Cascales, Eric and Pilhofer, Martin and Durand, Eric and Fronzes, Rémi},
title = {javax.xml.bind.JAXBElement@33b05f3, and high-resolution cryo-EM structure of a bacterial type VI secretion system membrane complex.},
journal = {The EMBO journal},
year = {2019},
volume = {38},
doi = {10.15252/embj.2018100886}
}
|
| Resch GP (2019), "Software for automated acquisition of electron tomography tilt series.", Methods in cell biology. Vol. 152, pp. 135-178. |
| Abstract: For automated acquisition of tilt series for electron tomography, software needs to handle complications such as movements of the sample in x/y and z, increased projected thickness at high tilt, specimen drift, etc. In addition, many applications require special functionality such as low dose acquisition, automated sequential (batch) tomography, or montage tomography. After reviewing how these difficulties can be addressed and a closer look at what advanced acquisition strategies are employed in biosciences, this chapter introduces acquisition software both developed in academia as well as by hardware vendors. It covers the hardware requirements and compatibility, the functional principle and workflow implemented, as well as what advanced functions are supported by the individual programs. |
BibTeX:
@article{Resch2019,
author = {Resch, Guenter P.},
title = {Software for automated acquisition of electron tomography tilt series.},
journal = {Methods in cell biology},
year = {2019},
volume = {152},
pages = {135--178},
doi = {10.1016/bs.mcb.2019.05.002}
}
|
| Reza M, Bertinetto C, Ruokolainen J and Vuorinen T (2017), "Cellulose Elementary Fibrils Assemble into Helical Bundles in S1 Layer of Spruce Tracheid Wall.", Biomacromolecules. Vol. 18, pp. 374-378. |
| Abstract: The ultrastructural organization of cellulose elementary fibrils (EFs) in wood cell wall is considered to be the prime factor regulating the material characteristics of wood in micro to macro levels and the conversion of delignified wood fibers into various products. Specifically, the complex assembly of EFs in wood cell wall limits its swellability, solubility, and reactivity, for example, in dissolution of cellulose for regeneration of textile fibers, fibril separation for the manufacture of nanocellulose, and enzymatic hydrolysis of cellulose into sugars for their subsequent fermentation to various products, like ethanol for future fossil fuels replacement. Here cryo-transmission electron tomography was applied on ultrathin spruce wood sections to reveal the EF assembly in S1 layer of the native cell wall. The resolution of these tomograms was then further enhanced by computational means. For the first time, cellulose in the intact cell wall was visualized to be assembled into helical bundles of several EFs, a structural feature that must have a significant impact on the swelling, accessibility, and solubility of woody biomass for its conversion into the aforementioned value added products. |
BibTeX:
@article{Reza2017,
author = {Reza, Mehedi and Bertinetto, Carlo and Ruokolainen, Janne and Vuorinen, Tapani},
title = {Cellulose Elementary Fibrils Assemble into Helical Bundles in S1 Layer of Spruce Tracheid Wall.},
journal = {Biomacromolecules},
year = {2017},
volume = {18},
pages = {374--378},
doi = {10.1021/acs.biomac.6b01396}
}
|
| Riedel C, Vasishtan D, Pražák V, Ghanem A, Conzelmann K-K and Rümenapf T (2019), "Cryo EM structure of the rabies virus ribonucleoprotein complex.", Scientific reports. Vol. 9, pp. 9639. |
| Abstract: Rabies virus is an important zoonotic pathogen. Its bullet shaped particle contains a helical nucleocapsid. We used cryo-electron tomography and subsequent subtomogram averaging to determine the structure of its ribonucleoprotein. The resulting electron density map allowed for confident fitting of the N-protein crystal structure, indicating that interactions between neighbouring N-proteins are only mediated by N- and C-terminal protruding subdomains (aa 1-27 and aa 355-372). Additional connecting densities, likely stabilizing the ribonucleoprotein complex, are present between neighbouring M-protein densities on the same helical turn and between M- and N-protein densities located on neighbouring helical turns, but not between M-proteins of different turns, as is observed for the related Vesicular stomatitis virus (VSV). This insight into the architecture of the rabies virus nucleocapsid highlights the surprising structural divergence of large biological assemblies even if the building blocks - here exemplified by VSV M- and N-protein - are structurally closely related. |
BibTeX:
@article{Riedel2019,
author = {Riedel, Christiane and Vasishtan, Daven and Pražák, Vojtěch and Ghanem, Alexander and Conzelmann, Karl-Klaus and Rümenapf, Till},
title = {Cryo EM structure of the rabies virus ribonucleoprotein complex.},
journal = {Scientific reports},
year = {2019},
volume = {9},
pages = {9639},
doi = {10.1038/s41598-019-46126-7}
}
|
| Riedel C, Vasishtan D, Siebert CA, Whittle C, Lehmann MJ, Mothes W and Grünewald K (2017), "Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell.", Journal of structural biology. Vol. 197, pp. 172-180. |
| Abstract: Enveloped viruses enter their host cells by membrane fusion. The process of attachment and fusion in retroviruses is mediated by a single viral envelope glycoprotein (Env). Conformational changes of Env in the course of fusion are a focus of intense studies. Here we provide further insight into the changes occurring in retroviral Env during its initial interaction with the cell, employing murine leukemia virus (MLV) as model system. We first determined the structure of both natively membrane anchored MLV Env and MLV Env tagged with YFP in the proline rich region (PRR) by electron cryo tomography (cET) and sub-volume averaging. At a resolution of ∼20Å, native MLV Env presents as a hollow trimer (height ∼85Å, diameter ∼120Å) composed of step-shaped protomers. The major difference to the YFP-tagged protein was in regions outside of the central trimer. Next, we focused on elucidating the changes in MLV Env upon interaction with a host cell. Virus interaction with the plasma membrane occurred over a large surface and Env clustering on the binding site was observed. Sub-volume averaging did yield a low-resolution structure of Env interacting with the cell, which had lost its threefold symmetry and was elongated by ∼35Å in comparison to the unbound protein. This indicates a major rearrangement of Env upon host cell binding. At the site of virus interaction, the otherwise clearly defined bilayer structure of the host cell plasma membrane was much less evident, indicative of integral membrane protein accumulation and/or a change in membrane lipid composition. |
BibTeX:
@article{Riedel2017,
author = {Riedel, Christiane and Vasishtan, Daven and Siebert, C Alistair and Whittle, Cathy and Lehmann, Maik J and Mothes, Walther and Grünewald, Kay},
title = {Native structure of a retroviral envelope protein and its conformational change upon interaction with the target cell.},
journal = {Journal of structural biology},
year = {2017},
volume = {197},
pages = {172--180},
doi = {10.1016/j.jsb.2016.06.017}
}
|
| Rossmann FM and Beeby M (2018), "Insights into the evolution of bacterial flagellar motors from high-throughput in situ electron cryotomography and subtomogram averaging.", Acta crystallographica. Section D, Structural biology. Vol. 74, pp. 585-594. |
| Abstract: In situ structural information on molecular machines can be invaluable in understanding their assembly, mechanism and evolution. Here, the use of electron cryotomography (ECT) to obtain significant insights into how an archetypal molecular machine, the bacterial flagellar motor, functions and how it has evolved is described. Over the last decade, studies using a high-throughput, medium-resolution ECT approach combined with genetics, phylogenetic reconstruction and phenotypic analysis have revealed surprising structural diversity in flagellar motors. Variations in the size and the number of torque-generating proteins in the motor visualized for the first time using ECT has shown that these variations have enabled bacteria to adapt their swimming torque to the environment. Much of the structural diversity can be explained in terms of scaffold structures that facilitate the incorporation of additional motor proteins, and more recent studies have begun to infer evolutionary pathways to higher torque-producing motors. This review seeks to highlight how the emerging power of ECT has enabled the inference of ancestral states from various bacterial species towards understanding how, and `why', flagellar motors have evolved from an ancestral motor to a diversity of variants with adapted or modified functions. |
BibTeX:
@article{Rossmann2018,
author = {Rossmann, Florian M and Beeby, Morgan},
title = {Insights into the evolution of bacterial flagellar motors from high-throughput in situ electron cryotomography and subtomogram averaging.},
journal = {Acta crystallographica. Section D, Structural biology},
year = {2018},
volume = {74},
pages = {585--594},
doi = {10.1107/S2059798318007945}
}
|
| Salzer R, D'Imprima E, Gold VAM, Rose I, Drechsler M, Vonck J and Averhoff B (2016), "Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus.", The Journal of biological chemistry. Vol. 291, pp. 14448-14456. |
| Abstract: Secretins are versatile outer membrane pores used by many bacteria to secrete proteins, toxins, or filamentous phages; extrude type IV pili (T4P); or take up DNA. Extrusion of T4P and natural transformation of DNA in the thermophilic bacterium Thermus thermophilus requires a unique secretin complex comprising six stacked rings, a membrane-embedded cone structure, and two gates that open and close a central channel. To investigate the role of distinct domains in ring and gate formation, we examined a set of deletion derivatives by cryomicroscopy techniques. Here we report that maintaining the N0 ring in the deletion derivatives led to stable PilQ complexes. Analyses of the variants unraveled that an N-terminal domain comprising a unique βββαβ fold is essential for the formation of gate 2. Furthermore, we identified four βαββα domains essential for the formation of the N2 to N5 rings. Mutant studies revealed that deletion of individual ring domains significantly reduces piliation. The N1, N2, N4, and N5 deletion mutants were significantly impaired in T4P-mediated twitching motility, whereas the motility of the N3 mutant was comparable with that of wild-type cells. This indicates that the deletion of the N3 ring leads to increased pilus dynamics, thereby compensating for the reduced number of pili of the N3 mutant. All mutants exhibit a wild-type natural transformation phenotype, leading to the conclusion that DNA uptake is independent of functional T4P. |
BibTeX:
@article{Salzer2016,
author = {Salzer, Ralf and D'Imprima, Edoardo and Gold, Vicki A M and Rose, Ilona and Drechsler, Moritz and Vonck, Janet and Averhoff, Beate},
title = {Topology and Structure/Function Correlation of Ring- and Gate-forming Domains in the Dynamic Secretin Complex of Thermus thermophilus.},
journal = {The Journal of biological chemistry},
year = {2016},
volume = {291},
pages = {14448--14456},
doi = {10.1074/jbc.M116.724153}
}
|
| Scaramuzza S and Castaño-Díez D (2021), "Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo.", PLoS biology. Vol. 19, pp. e3001318. |
| Abstract: Subtomogram averaging (STA) is a powerful image processing technique in electron tomography used to determine the 3D structure of macromolecular complexes in their native environments. It is a fast growing technique with increasing importance in structural biology. The computational aspect of STA is very complex and depends on a large number of variables. We noticed a lack of detailed guides for STA processing. Also, current publications in this field often lack a documentation that is practical enough to reproduce the results with reasonable effort, which is necessary for the scientific community to grow. We therefore provide a complete, detailed, and fully reproducible processing protocol that covers all aspects of particle picking and particle alignment in STA. The command line-based workflow is fully based on the popular Dynamo software for STA. Within this workflow, we also demonstrate how large parts of the processing pipeline can be streamlined and automatized for increased throughput. This protocol is aimed at users on all levels. It can be used for training purposes, or it can serve as basis to design user-specific projects by taking advantage of the flexibility of Dynamo by modifying and expanding the given pipeline. The protocol is successfully validated using the Electron Microscopy Public Image Archive (EMPIAR) database entry 10164 from immature HIV-1 virus-like particles (VLPs) that describe a geometry often seen in electron tomography. |
BibTeX:
@article{Scaramuzza2021,
author = {Scaramuzza, Stefano and Castaño-Díez, Daniel},
title = {Step-by-step guide to efficient subtomogram averaging of virus-like particles with Dynamo.},
journal = {PLoS biology},
year = {2021},
volume = {19},
pages = {e3001318},
doi = {10.1371/journal.pbio.3001318}
}
|
| Scharf L, Wang H, Gao H, Chen S, McDowall AW and Bjorkman PJ (2015), "Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.", Cell. Vol. 162(6), pp. 1379-1390. |
| Abstract: The HIV-1 envelope (Env) spike contains limited epitopes for broadly neutralizing antibodies (bNAbs); thus, most neutralizing antibodies are strain specific. The 8ANC195 epitope, defined by crystal and electron microscopy (EM) structures of bNAb 8ANC195 complexed with monomeric gp120 and trimeric Env, respectively, spans the gp120 and gp41 Env subunits. To investigate 8ANC195's gp41 epitope at higher resolution, we solved a 3.58 Å crystal structure of 8ANC195 complexed with fully glycosylated Env trimer, revealing 8ANC195 insertion into a glycan shield gap to contact gp120 and gp41 glycans and protein residues. To determine whether 8ANC195 recognizes the CD4-bound open Env conformation that leads to co-receptor binding and fusion, one of several known conformations of virion-associated Env, we solved EM structures of an Env/CD4/CD4-induced antibody/8ANC195 complex. 8ANC195 binding partially closed the CD4-bound trimer, confirming structural plasticity of Env by revealing a previously unseen conformation. 8ANC195's ability to bind different Env conformations suggests advantages for potential therapeutic applications. |
BibTeX:
@article{Scharf2015,
author = {Scharf, Louise and Wang, Haoqing and Gao, Han and Chen, Songye and McDowall, Alasdair W. and Bjorkman, Pamela J.},
title = {Broadly Neutralizing Antibody 8ANC195 Recognizes Closed and Open States of HIV-1 Env.},
journal = {Cell},
school = {Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA. Electronic address: bjorkman@caltech.edu.},
year = {2015},
volume = {162},
number = {6},
pages = {1379--1390},
url = {http://dx.doi.org/10.1016/j.cell.2015.08.035},
doi = {10.1016/j.cell.2015.08.035}
}
|
| Scheres SHW (2016), "Processing of Structurally Heterogeneous Cryo-EM Data in RELION.", Methods in enzymology. Vol. 579, pp. 125-157. |
| Abstract: This chapter describes algorithmic advances in the RELION software, and how these are used in high-resolution cryo-electron microscopy (cryo-EM) structure determination. Since the presence of projections of different three-dimensional structures in the dataset probably represents the biggest challenge in cryo-EM data processing, special emphasis is placed on how to deal with structurally heterogeneous datasets. As such, this chapter aims to be of practical help to those who wish to use RELION in their cryo-EM structure determination efforts. |
BibTeX:
@article{Scheres2016,
author = {Scheres, S H W},
title = {Processing of Structurally Heterogeneous Cryo-EM Data in RELION.},
journal = {Methods in enzymology},
year = {2016},
volume = {579},
pages = {125--157},
doi = {10.1016/bs.mie.2016.04.012}
}
|
| Schwartz CL, Heumann JM, Dawson SC and Hoenger A (2012), "A detailed, hierarchical study of Giardia lamblia's ventral disc reveals novel microtubule-associated protein complexes.", PLoS One. Vol. 7(9), pp. e43783. |
| Abstract: Giardia lamblia is a flagellated, unicellular parasite of mammals infecting over one billion people worldwide. Giardia's two-stage life cycle includes a motile trophozoite stage that colonizes the host small intestine and an infectious cyst form that can persist in the environment. Similar to many eukaryotic cells, Giardia contains several complex microtubule arrays that are involved in motility, chromosome segregation, organelle transport, maintenance of cell shape and transformation between the two life cycle stages. Giardia trophozoites also possess a unique spiral microtubule array, the ventral disc, made of approximately 50 parallel microtubules and associated microribbons, as well as a variety of associated proteins. The ventral disc maintains trophozoite attachment to the host intestinal epithelium. With the help of a combined SEM/microtome based slice and view method called 3View® (Gatan Inc., Pleasanton, CA), we present an entire trophozoite cell reconstruction and describe the arrangement of the major cytoskeletal elements. To aid in future analyses of disc-mediated attachment, we used electron-tomography of freeze-substituted, plastic-embedded trophozoites to explore the detailed architecture of ventral disc microtubules and their associated components. Lastly, we examined the disc microtubule array in three dimensions in unprecedented detail using cryo-electron tomography combined with internal sub-tomogram volume averaging of repetitive domains. We discovered details of protein complexes stabilizing microtubules by attachment to their inner and outer wall. A unique tri-laminar microribbon structure is attached vertically to the disc microtubules and is connected to neighboring microribbons via crossbridges. This work provides novel insight into the structure of the ventral disc microtubules, microribbons and associated proteins. Knowledge of the components comprising these structures and their three-dimensional organization is crucial toward understanding how attachment via the ventral disc occurs in vivo. |
BibTeX:
@article{Schwartz2012,
author = {Schwartz, Cindi L. and Heumann, John M. and Dawson, Scott C. and Hoenger, Andreas},
title = {A detailed, hierarchical study of Giardia lamblia's ventral disc reveals novel microtubule-associated protein complexes.},
journal = {PLoS One},
school = {Boulder Lab for 3-D Electron Microscopy of Cells, Department of MCD Biology, University of Colorado, Boulder, Colorado, United States of America.},
year = {2012},
volume = {7},
number = {9},
pages = {e43783},
url = {http://dx.doi.org/10.1371/journal.pone.0043783},
doi = {10.1371/journal.pone.0043783}
}
|
| Shimogawa MM, Wijono AS, Wang H, Zhang J, Sha J, Szombathy N, Vadakkan S, Pelayo P, Jonnalagadda K, Wohlschlegel J, Zhou ZH and Hill KL (), "FAP106 is an interaction hub required for assembly of conserved and lineage-specific microtubule inner proteins at the cilium inner junction". |
BibTeX:
@misc{Shimogawa,
author = {Michelle M. Shimogawa and Angeline S. Wijono and Hui Wang and Jiayan Zhang and Jihui Sha and Natasha Szombathy and Sabeeca Vadakkan and Paula Pelayo and Keya Jonnalagadda and James Wohlschlegel and Z. Hong Zhou and Kent L. Hill},
title = {FAP106 is an interaction hub required for assembly of conserved and lineage-specific microtubule inner proteins at the cilium inner junction},
doi = {10.1101/2022.11.11.516029}
}
|
| Si Z, Zhang J, Shivakoti S, Atanasov I, Tao C-L, Hui WH, Zhou K, Yu X, Li W, Luo M, Bi G-Q and Zhou ZH (2018), "Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate.", PLoS pathogens. Vol. 14, pp. e1007452. |
| Abstract: Human cytomegalovirus (HCMV) enters host by glycoprotein B (gB)-mediated membrane fusion upon receptor-binding to gH/gL-related complexes, causing devastating diseases such as birth defects. Although an X-ray crystal structure of the recombinant gB ectodomain at postfusion conformation is available, the structures of prefusion gB and its complex with gH/gL on the viral envelope remain elusive. Here, we demonstrate the utility of cryo electron tomography (cryoET) with energy filtering and the cutting-edge technologies of Volta phase plate (VPP) and direct electron-counting detection to capture metastable prefusion viral fusion proteins and report the structures of glycoproteins in the native environment of HCMV virions. We established the validity of our approach by obtaining cryoET in situ structures of the vesicular stomatitis virus (VSV) glycoprotein G trimer (171 kD) in prefusion and postfusion conformations, which agree with the known crystal structures of purified G trimers in both conformations. The excellent contrast afforded by these technologies has enabled us to identify gB trimers (303kD) in two distinct conformations in HCMV tomograms and obtain their in situ structures at up to 21 Å resolution through subtomographic averaging. The predominant conformation (79%), which we designate as gB prefusion conformation, fashions a globular endodomain and a Christmas tree-shaped ectodomain, while the minority conformation (21%) has a columnar tree-shaped ectodomain that matches the crystal structure of the "postfusion" gB ectodomain. We also observed prefusion gB in complex with an "L"-shaped density attributed to the gH/gL complex. Integration of these structures of HCMV glycoproteins in multiple functional states and oligomeric forms with existing biochemical data and domain organization of other class III viral fusion proteins suggests that gH/gL receptor-binding triggers conformational changes of gB endodomain, which in turn triggers two essential steps to actuate virus-cell membrane fusion: exposure of gB fusion loops and unfurling of gB ectodomain. |
BibTeX:
@article{Si2018,
author = {Si, Zhu and Zhang, Jiayan and Shivakoti, Sakar and Atanasov, Ivo and Tao, Chang-Lu and Hui, Wong H and Zhou, Kang and Yu, Xuekui and Li, Weike and Luo, Ming and Bi, Guo-Qiang and Zhou, Z Hong},
title = {Different functional states of fusion protein gB revealed on human cytomegalovirus by cryo electron tomography with Volta phase plate.},
journal = {PLoS pathogens},
year = {2018},
volume = {14},
pages = {e1007452},
doi = {10.1371/journal.ppat.1007452}
}
|
| Silvester E, Vollmer B, Pražák V, Vasishtan D, Machala EA, Whittle C, Black S, Bath J, Turberfield AJ, Grünewald K and Baker LA (2021), "DNA origami signposts for identifying proteins on cell membranes by electron cryotomography.", Cell. Vol. 184, pp. 1110-1121.e16. |
| Abstract: Electron cryotomography (cryoET), an electron cryomicroscopy (cryoEM) modality, has changed our understanding of biological function by revealing the native molecular details of membranes, viruses, and cells. However, identification of individual molecules within tomograms from cryoET is challenging because of sample crowding and low signal-to-noise ratios. Here, we present a tagging strategy for cryoET that precisely identifies individual protein complexes in tomograms without relying on metal clusters. Our method makes use of DNA origami to produce "molecular signposts" that target molecules of interest, here via fluorescent fusion proteins, providing a platform generally applicable to biological surfaces. We demonstrate the specificity of signpost origami tags (SPOTs) in vitro as well as their suitability for cryoET of membrane vesicles, enveloped viruses, and the exterior of intact mammalian cells. |
BibTeX:
@article{Silvester2021,
author = {Silvester, Emma and Vollmer, Benjamin and Pražák, Vojtěch and Vasishtan, Daven and Machala, Emily A. and Whittle, Catheryne and Black, Susan and Bath, Jonathan and Turberfield, Andrew J. and Grünewald, Kay and Baker, Lindsay A.},
title = {DNA origami signposts for identifying proteins on cell membranes by electron cryotomography.},
journal = {Cell},
year = {2021},
volume = {184},
pages = {1110--1121.e16},
doi = {10.1016/j.cell.2021.01.033}
}
|
| Song K, Awata J, Tritschler D, Bower R, Witman GB, Porter ME and Nicastro D (2015), "In situ localization of N and C termini of subunits of the flagellar nexin-dynein regulatory complex (N-DRC) using SNAP tag and cryo-electron tomography.", J Biol Chem. Vol. 290(9), pp. 5341-5353. |
| Abstract: Cryo-electron tomography (cryo-ET) has reached nanoscale resolution for in situ three-dimensional imaging of macromolecular complexes and organelles. Yet its current resolution is not sufficient to precisely localize or identify most proteins in situ; for example, the location and arrangement of components of the nexin-dynein regulatory complex (N-DRC), a key regulator of ciliary/flagellar motility that is conserved from algae to humans, have remained elusive despite many cryo-ET studies of cilia and flagella. Here, we developed an in situ localization method that combines cryo-ET/subtomogram averaging with the clonable SNAP tag, a widely used cell biological probe to visualize fusion proteins by fluorescence microscopy. Using this hybrid approach, we precisely determined the locations of the N and C termini of DRC3 and the C terminus of DRC4 within the three-dimensional structure of the N-DRC in Chlamydomonas flagella. Our data demonstrate that fusion of SNAP with target proteins allowed for protein localization with high efficiency and fidelity using SNAP-linked gold nanoparticles, without disrupting the native assembly, structure, or function of the flagella. After cryo-ET and subtomogram averaging, we localized DRC3 to the L1 projection of the nexin linker, which interacts directly with a dynein motor, whereas DRC4 was observed to stretch along the N-DRC base plate to the nexin linker. Application of the technique developed here to the N-DRC revealed new insights into the organization and regulatory mechanism of this complex, and provides a valuable tool for the structural dissection of macromolecular complexes in situ. |
BibTeX:
@article{Song2015,
author = {Song, Kangkang and Awata, Junya and Tritschler, Douglas and Bower, Raqual and Witman, George B. and Porter, Mary E. and Nicastro, Daniela},
title = {In situ localization of N and C termini of subunits of the flagellar nexin-dynein regulatory complex (N-DRC) using SNAP tag and cryo-electron tomography.},
journal = {J Biol Chem},
school = {From the Biology Department, Brandeis University, Waltham, Massachusetts 02454, nicastro@brandeis.edu.},
year = {2015},
volume = {290},
number = {9},
pages = {5341--5353},
url = {http://dx.doi.org/10.1074/jbc.M114.626556},
doi = {10.1074/jbc.M114.626556}
}
|
| Sparvoli D, Delabre J, Penarete‐Vargas DM, Mageswaran SK, Tsypin LM, Heckendorn J, Theveny L, Maynadier M, Cova MM, Berry‐Sterkers L, Guérin A, Dubremetz J, Urbach S, Striepen B, Turkewitz AP, Chang Y and Lebrun M (2022), "An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma" Vol. 41 |
BibTeX:
@article{Sparvoli2022,
author = {Daniela Sparvoli and Jason Delabre and Diana Marcela Penarete‐Vargas and Shrawan Kumar Mageswaran and Lev M. Tsypin and Justine Heckendorn and Liam Theveny and Marjorie Maynadier and Marta Mendonça Cova and Laurence Berry‐Sterkers and Amandine Guérin and Jean‐François Dubremetz and Serge Urbach and Boris Striepen and Aaron P. Turkewitz and Yi‐Wei Chang and Maryse Lebrun},
title = {An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma},
year = {2022},
volume = {41},
doi = {10.15252/embj.2022111158}
}
|
| Stobart CC, Rostad CA, Ke Z, Dillard RS, Hampton CM, Strauss JD, Yi H, Hotard AL, Meng J, Pickles RJ, Sakamoto K, Lee S, Currier MG, Moin SM, Graham BS, Boukhvalova MS, Gilbert BE, Blanco JCG, Piedra PA, Wright ER and Moore ML (2016), "A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation.", Nature communications. Vol. 7, pp. 13916. |
| Abstract: Respiratory syncytial virus (RSV) is a leading cause of infant hospitalization and there remains no pediatric vaccine. RSV live-attenuated vaccines (LAVs) have a history of safe testing in infants; however, achieving an effective balance of attenuation and immunogenicity has proven challenging. Here we seek to engineer an RSV LAV with enhanced immunogenicity. Genetic mapping identifies strain line 19 fusion (F) protein residues that correlate with pre-fusion antigen maintenance by ELISA and thermal stability of infectivity in live RSV. We generate a LAV candidate named OE4 which expresses line 19F and is attenuated by codon-deoptimization of non-structural (NS1 and NS2) genes, deletion of the small hydrophobic (SH) gene, codon-deoptimization of the attachment (G) gene and ablation of the secreted form of G. OE4 (RSV-A2-dNS1-dNS2-ΔSH-dGm-Gsnull-line19F) exhibits elevated pre-fusion antigen levels, thermal stability, immunogenicity, and efficacy despite heavy attenuation in the upper and lower airways of cotton rats. |
BibTeX:
@article{Stobart2016a,
author = {Stobart, Christopher C and Rostad, Christina A and Ke, Zunlong and Dillard, Rebecca S and Hampton, Cheri M and Strauss, Joshua D and Yi, Hong and Hotard, Anne L and Meng, Jia and Pickles, Raymond J and Sakamoto, Kaori and Lee, Sujin and Currier, Michael G and Moin, Syed M and Graham, Barney S and Boukhvalova, Marina S and Gilbert, Brian E and Blanco, Jorge C G and Piedra, Pedro A and Wright, Elizabeth R and Moore, Martin L},
title = {A live RSV vaccine with engineered thermostability is immunogenic in cotton rats despite high attenuation.},
journal = {Nature communications},
year = {2016},
volume = {7},
pages = {13916},
doi = {10.1038/ncomms13916}
}
|
| Stoddard D, Zhao Y, Bayless BA, Gui L, Louka P, Dave D, Suryawanshi S, Tomasi RF-X, Dupuis-Williams P, Baroud CN, Gaertig J, Winey M and Nicastro D (2018), "Tetrahymena RIB72A and RIB72B are microtubule inner proteins in the ciliary doublet microtubules.", Molecular biology of the cell. Vol. 29, pp. 2566-2577. |
| Abstract: Doublet and triplet microtubules are essential and highly stable core structures of centrioles, basal bodies, cilia, and flagella. In contrast to dynamic cytoplasmic micro-tubules, their luminal surface is coated with regularly arranged microtubule inner proteins (MIPs). However, the protein composition and biological function(s) of MIPs remain poorly understood. Using genetic, biochemical, and imaging techniques, we identified Tetrahymena RIB72A and RIB72B proteins as ciliary MIPs. Fluorescence imaging of tagged RIB72A and RIB72B showed that both proteins colocalize to Tetrahymena cilia and basal bodies but assemble independently. Cryoelectron tomography of RIB72A and/or RIB72B knockout strains revealed major structural defects in the ciliary A-tubule involving MIP1, MIP4, and MIP6 structures. The defects of individual mutants were complementary in the double mutant. All mutants had reduced swimming speed and ciliary beat frequencies, and high-speed video imaging revealed abnormal highly curved cilia during power stroke. Our results show that RIB72A and RIB72B are crucial for the structural assembly of ciliary A-tubule MIPs and are important for proper ciliary motility. |
BibTeX:
@article{Stoddard2018,
author = {Stoddard, Daniel and Zhao, Ying and Bayless, Brian A and Gui, Long and Louka, Panagiota and Dave, Drashti and Suryawanshi, Swati and Tomasi, Raphaël F-X and Dupuis-Williams, Pascale and Baroud, Charles N and Gaertig, Jacek and Winey, Mark and Nicastro, Daniela},
title = {Tetrahymena RIB72A and RIB72B are microtubule inner proteins in the ciliary doublet microtubules.},
journal = {Molecular biology of the cell},
year = {2018},
volume = {29},
pages = {2566--2577},
doi = {10.1091/mbc.E18-06-0405}
}
|
| Strauss M, Levy HC, Bostina M, Filman DJ and Hogle JM (2013), "RNA transfer from poliovirus 135S particles across membranes is mediated by long umbilical connectors.", J Virol. Vol. 87(7), pp. 3903-3914. |
| Abstract: During infection, the binding of poliovirus to its cell surface receptor at 37°C triggers an expansion of the virus in which internal polypeptides that bind to membranes are externalized. Subsequently, in a poorly understood process, the viral RNA genome is transferred directly across an endosomal membrane, and into the host cell cytoplasm, to initiate infection. Here, cryoelectron tomography demonstrates the results of 37°C warming of a poliovirus-receptor-liposome model complex that was produced using Ni-nitrilotriacetic acid lipids and His-tagged receptor ectodomains. In total, 651 subtomographic volumes were aligned, classified, and averaged to obtain detailed pictures, showing both the conversion of virus into its expanded form and the passage of RNA into intact liposomes. Unexpectedly, the virus and membrane surfaces were located ∼50 Å apart, with the 5-fold axis tilted away from the perpendicular, and the solvent spaces between them were spanned by either one or two long "umbilical" density features that lie at an angle to the virus and membrane. The thinner connector, which sometimes appears alone, is 28 to 30 Å in diameter and has a footprint on the virus surface located close to either a 5-fold or a 3-fold axis. The broader connector has a footprint near the quasi-3-fold hole that opens upon virus expansion and is hypothesized to include RNA, shielded from enzymatic degradation by polypeptides that include the N-terminal extension of VP1 and capsid protein VP4. The implications of these observations for the mechanism of RNase-protected RNA transfer in picornaviruses are discussed. |
BibTeX:
@article{Strauss2013,
author = {Strauss, Mike and Levy, Hazel C. and Bostina, Mihnea and Filman, David J. and Hogle, James M.},
title = {RNA transfer from poliovirus 135S particles across membranes is mediated by long umbilical connectors.},
journal = {J Virol},
school = {Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts, USA.},
year = {2013},
volume = {87},
number = {7},
pages = {3903--3914},
url = {http://dx.doi.org/10.1128/JVI.03209-12},
doi = {10.1128/JVI.03209-12}
}
|
| Syrjänen JL, Heller I, Candelli A, Davies OR, Peterman EJG, Wuite GJL and Pellegrini L (2017), "Single-molecule observation of DNA compaction by meiotic protein SYCP3.", eLife. Vol. 6 |
| Abstract: In a previous paper (Syrjänen et al., 2014), we reported the first structural characterisation of a synaptonemal complex (SC) protein, SYCP3, which led us to propose a model for its role in chromosome compaction during meiosis. As a component of the SC lateral element, SYCP3 has a critical role in defining the specific chromosome architecture required for correct meiotic progression. In the model, the reported compaction of chromosomal DNA caused by SYCP3 would result from its ability to bridge distant sites on a DNA molecule with the DNA-binding domains located at each end of its strut-like structure. Here, we describe a single-molecule assay based on optical tweezers, fluorescence microscopy and microfluidics that, in combination with bulk biochemical data, provides direct visual evidence for our proposed mechanism of SYCP3-mediated chromosome organisation. |
BibTeX:
@article{Syrjaenen2017,
author = {Syrjänen, Johanna L and Heller, Iddo and Candelli, Andrea and Davies, Owen R and Peterman, Erwin J G and Wuite, Gijs J L and Pellegrini, Luca},
title = {Single-molecule observation of DNA compaction by meiotic protein SYCP3.},
journal = {eLife},
year = {2017},
volume = {6},
doi = {10.7554/eLife.22582}
}
|
| Syrjänen JL, Pellegrini L and Davies OR (2014), "A molecular model for the role of SYCP3 in meiotic chromosome organisation.", eLife. Vol. 3 |
| Abstract: The synaptonemal complex (SC) is an evolutionarily-conserved protein assembly that holds together homologous chromosomes during prophase of the first meiotic division. Whilst essential for meiosis and fertility, the molecular structure of the SC has proved resistant to elucidation. The SC protein SYCP3 has a crucial but poorly understood role in establishing the architecture of the meiotic chromosome. Here we show that human SYCP3 forms a highly-elongated helical tetramer of 20 nm length. N-terminal sequences extending from each end of the rod-like structure bind double-stranded DNA, enabling SYCP3 to link distant sites along the sister chromatid. We further find that SYCP3 self-assembles into regular filamentous structures that resemble the known morphology of the SC lateral element. Together, our data form the basis for a model in which SYCP3 binding and assembly on meiotic chromosomes leads to their organisation into compact structures compatible with recombination and crossover formation. |
BibTeX:
@article{Syrjaenen2014,
author = {Syrjänen, Johanna Liinamaria and Pellegrini, Luca and Davies, Owen Richard},
title = {A molecular model for the role of SYCP3 in meiotic chromosome organisation.},
journal = {eLife},
year = {2014},
volume = {3},
doi = {10.7554/eLife.02963}
}
|
| Tero T-R, Malola S, Koncz B, Pohjolainen E, Lautala S, Mustalahti S, Permi P, Groenhof G, Pettersson M and Häkkinen H (2017), "Dynamic stabilization of the ligand--metal interface in atomically precise gold nanoclusters Au68 and Au144 protected by meta-mercaptobenzoic acid", ACS nano. Vol. 11(12), pp. 11872-11879. ACS Publications.
[BibTeX] |
BibTeX:
@article{Tero2017,
author = {Tero, Tiia-Riikka and Malola, Sami and Koncz, Benedek and Pohjolainen, Emmi and Lautala, Saara and Mustalahti, Satu and Permi, Perttu and Groenhof, Gerrit and Pettersson, Mika and Häkkinen, Hannu},
title = {Dynamic stabilization of the ligand--metal interface in atomically precise gold nanoclusters Au68 and Au144 protected by meta-mercaptobenzoic acid},
journal = {ACS nano},
publisher = {ACS Publications},
year = {2017},
volume = {11},
number = {12},
pages = {11872--11879}
}
|
| Turk M and Baumeister W (2020), "The promise and the challenges of cryo-electron tomography.", FEBS letters. Vol. 594, pp. 3243-3261. |
| Abstract: Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This 'divide and conquer' approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo-electron tomography (Cryo-ET) combines the power of 3D molecular-level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or 'molecular sociology' of cells and to discover the unexpected. Here, we review state-of-the-art Cryo-ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo-ET. |
BibTeX:
@article{Turk2020,
author = {Turk, Martin and Baumeister, Wolfgang},
title = {The promise and the challenges of cryo-electron tomography.},
journal = {FEBS letters},
year = {2020},
volume = {594},
pages = {3243--3261},
doi = {10.1002/1873-3468.13948}
}
|
| Unchwaniwala N, Zhan H, Pennington J, Horswill M, den Boon JA and Ahlquist P (2020), "Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication.", Proceedings of the National Academy of Sciences of the United States of America. Vol. 117, pp. 18680-18691. |
| Abstract: For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter "crown" complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ∼19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ∼35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis. |
BibTeX:
@article{Unchwaniwala2020,
author = {Unchwaniwala, Nuruddin and Zhan, Hong and Pennington, Janice and Horswill, Mark and den Boon, Johan A. and Ahlquist, Paul},
title = {Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication.},
journal = {Proceedings of the National Academy of Sciences of the United States of America},
year = {2020},
volume = {117},
pages = {18680--18691},
doi = {10.1073/pnas.2006165117}
}
|
| Urbanska P, Song K, Joachimiak E, Krzemien-Ojak L, Koprowski P, Hennessey T, Jerka-Dziadosz M, Fabczak H, Gaertig J, Nicastro D and Wloga D (2015), "The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia.", Mol Biol Cell. Vol. 26(8), pp. 1463-1475. |
| Abstract: Dynein motors and regulatory complexes repeat every 96 nm along the length of motile cilia. Each repeat contains three radial spokes, RS1, RS2, and RS3, which transduct signals between the central microtubules and dynein arms. Each radial spoke has a distinct structure, but little is known about the mechanisms of assembly and function of the individual radial spokes. In Chlamydomonas, calmodulin and spoke-associated complex (CSC) is composed of FAP61, FAP91, and FAP251 and has been linked to the base of RS2 and RS3. We show that in Tetrahymena, loss of either FAP61 or FAP251 reduces cell swimming and affects the ciliary waveform and that RS3 is either missing or incomplete, whereas RS1 and RS2 are unaffected. Specifically, FAP251-null cilia lack an arch-like density at the RS3 base, whereas FAP61-null cilia lack an adjacent portion of the RS3 stem region. This suggests that the CSC proteins are crucial for stable and functional assembly of RS3 and that RS3 and the CSC are important for ciliary motility. |
BibTeX:
@article{Urbanska2015,
author = {Urbanska, Paulina and Song, Kangkang and Joachimiak, Ewa and Krzemien-Ojak, Lucja and Koprowski, Piotr and Hennessey, Todd and Jerka-Dziadosz, Maria and Fabczak, Hanna and Gaertig, Jacek and Nicastro, Daniela and Wloga, Dorota},
title = {The CSC proteins FAP61 and FAP251 build the basal substructures of radial spoke 3 in cilia.},
journal = {Mol Biol Cell},
school = {Department of Cell Biology, Nencki Institute of Experimental Biology PAS, 02-093 Warsaw, Poland dwloga@nencki.gov.pl nicastro@brandeis.edu.},
year = {2015},
volume = {26},
number = {8},
pages = {1463--1475},
url = {http://dx.doi.org/10.1091/mbc.E14-11-1545},
doi = {10.1091/mbc.E14-11-1545}
}
|
| Vasudevan KK, Song K, Alford LM, Sale WS, Dymek EE, Smith EF, Hennessey T, Joachimiak E, Urbanska P, Wloga D, Dentler W, Nicastro D and Gaertig J (2015), "FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.", Mol Biol Cell. Vol. 26(4), pp. 696-710. |
| Abstract: Radial spokes are conserved macromolecular complexes that are essential for ciliary motility. A triplet of three radial spokes, RS1, RS2, and RS3, repeats every 96 nm along the doublet microtubules. Each spoke has a distinct base that docks to the doublet and is linked to different inner dynein arms. Little is known about the assembly and functions of individual radial spokes. A knockout of the conserved ciliary protein FAP206 in the ciliate Tetrahymena resulted in slow cell motility. Cryo-electron tomography showed that in the absence of FAP206, the 96-nm repeats lacked RS2 and dynein c. Occasionally, RS2 assembled but lacked both the front prong of its microtubule base and dynein c, whose tail is attached to the front prong. Overexpressed GFP-FAP206 decorated nonciliary microtubules in vivo. Thus FAP206 is likely part of the front prong and docks RS2 and dynein c to the microtubule. |
BibTeX:
@article{Vasudevan2015,
author = {Vasudevan, Krishna Kumar and Song, Kangkang and Alford, Lea M. and Sale, Winfield S. and Dymek, Erin E. and Smith, Elizabeth F. and Hennessey, Todd and Joachimiak, Ewa and Urbanska, Paulina and Wloga, Dorota and Dentler, William and Nicastro, Daniela and Gaertig, Jacek},
title = {FAP206 is a microtubule-docking adapter for ciliary radial spoke 2 and dynein c.},
journal = {Mol Biol Cell},
school = {Department of Cellular Biology, University of Georgia, Athens, GA 30602.},
year = {2015},
volume = {26},
number = {4},
pages = {696--710},
url = {http://dx.doi.org/10.1091/mbc.E14-11-1506},
doi = {10.1091/mbc.E14-11-1506}
}
|
| Wang J and others (2020), "Cryo-ET Workflow for Understanding Ion Channels Localization on the Nodes of Ranvier", Journal of Biosciences and Medicines. Vol. 8(02), pp. 55. Scientific Research Publishing.
[BibTeX] |
BibTeX:
@article{Wang2020,
author = {Wang, Jiaxuan and others},
title = {Cryo-ET Workflow for Understanding Ion Channels Localization on the Nodes of Ranvier},
journal = {Journal of Biosciences and Medicines},
publisher = {Scientific Research Publishing},
year = {2020},
volume = {8},
number = {02},
pages = {55}
}
|
| Wang Z, Grange M, Wagner T, Kho AL, Gautel M and Raunser S (2021), "The molecular basis for sarcomere organization in vertebrate skeletal muscle.", Cell. Vol. 184, pp. 2135-2150.e13. |
| Abstract: Sarcomeres are force-generating and load-bearing devices of muscles. A precise molecular picture of how sarcomeres are built underpins understanding their role in health and disease. Here, we determine the molecular architecture of native vertebrate skeletal sarcomeres by electron cryo-tomography. Our reconstruction reveals molecular details of the three-dimensional organization and interaction of actin and myosin in the A-band, I-band, and Z-disc and demonstrates that α-actinin cross-links antiparallel actin filaments by forming doublets with 6-nm spacing. Structures of myosin, tropomyosin, and actin at 10 Å further reveal two conformations of the "double-head" myosin, where the flexible orientation of the lever arm and light chains enable myosin not only to interact with the same actin filament, but also to split between two actin filaments. Our results provide unexpected insights into the fundamental organization of vertebrate skeletal muscle and serve as a strong foundation for future investigations of muscle diseases. |
BibTeX:
@article{Wang2021,
author = {Wang, Zhexin and Grange, Michael and Wagner, Thorsten and Kho, Ay Lin and Gautel, Mathias and Raunser, Stefan},
title = {The molecular basis for sarcomere organization in vertebrate skeletal muscle.},
journal = {Cell},
year = {2021},
volume = {184},
pages = {2135--2150.e13},
doi = {10.1016/j.cell.2021.02.047}
}
|
| Wang Z, Zhang Q and Mim C (2021), "Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces.", International journal of molecular sciences. Vol. 22 |
| Abstract: Over the last few years, cryo electron microscopy has become the most important method in structural biology. While 80% of deposited maps are from single particle analysis, electron tomography has grown to become the second most important method. In particular sub-tomogram averaging has matured as a method, delivering structures between 2 and 5 Å from complexes in cells as well as in vitro complexes. While this resolution range is not standard, novel developments point toward a promising future. Here, we provide a guide for the workflow from sample to structure to gain insight into this emerging field. |
BibTeX:
@article{Wang2021a,
author = {Wang, Zuoneng and Zhang, Qingyang and Mim, Carsten},
title = {Coming of Age: Cryo-Electron Tomography as a Versatile Tool to Generate High-Resolution Structures at Cellular/Biological Interfaces.},
journal = {International journal of molecular sciences},
year = {2021},
volume = {22},
doi = {10.3390/ijms22126177}
}
|
| Weiss GL, Eisenstein F, Kieninger A-K, Xu J, Minas HA, Gerber M, Feldmüller M, Maldener I, Forchhammer K and Pilhofer M (), "Structure of a thylakoid-anchored contractile injection system in multicellular cyanobacteria" Vol. 7, pp. 386-396. |
BibTeX:
@article{Weiss,
author = {Gregor L. Weiss and Fabian Eisenstein and Ann-Katrin Kieninger and Jingwei Xu and Hannah A. Minas and Milena Gerber and Miki Feldmüller and Iris Maldener and Karl Forchhammer and Martin Pilhofer},
title = {Structure of a thylakoid-anchored contractile injection system in multicellular cyanobacteria},
volume = {7},
pages = {386-396},
doi = {10.1038/s41564-021-01055-y}
}
|
| Weiss GL, Kieninger A-K, Maldener I, Forchhammer K and Pilhofer M (2019), "Structure and Function of a Bacterial Gap Junction Analog.", Cell. Vol. 178, pp. 374-384.e15. |
| Abstract: Multicellular lifestyle requires cell-cell connections. In multicellular cyanobacteria, septal junctions enable molecular exchange between sister cells and are required for cellular differentiation. The structure of septal junctions is poorly understood, and it is unknown whether they are capable of controlling intercellular communication. Here, we resolved the in situ architecture of septal junctions by electron cryotomography of cryo-focused ion beam-milled cyanobacterial filaments. Septal junctions consisted of a tube traversing the septal peptidoglycan. Each tube end comprised a FraD-containing plug, which was covered by a cytoplasmic cap. Fluorescence recovery after photobleaching showed that intercellular communication was blocked upon stress. Gating was accompanied by a reversible conformational change of the septal junction cap. We provide the mechanistic framework for a cell junction that predates eukaryotic gap junctions by a billion years. The conservation of a gated dynamic mechanism across different domains of life emphasizes the importance of controlling molecular exchange in multicellular organisms. |
BibTeX:
@article{Weiss2019,
author = {Weiss, Gregor L and Kieninger, Ann-Katrin and Maldener, Iris and Forchhammer, Karl and Pilhofer, Martin},
title = {Structure and Function of a Bacterial Gap Junction Analog.},
journal = {Cell},
year = {2019},
volume = {178},
pages = {374--384.e15},
doi = {10.1016/j.cell.2019.05.055}
}
|
| Weiss GL, Medeiros JM and Pilhofer M (2017), "In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography.", Methods in molecular biology (Clifton, N.J.). Vol. 1615, pp. 353-375. |
| Abstract: The unique property of electron cryotomography (ECT) is its capability to resolve the structure of macromolecular machines in their cellular context. The integration of ECT data with high-resolution structures of purified subcomplexes and live-cell fluorescence light microscopy can generate pseudo-atomic models that lead to a mechanistic understanding across size and time scales. Recent advances in electron detection, sample thinning, data acquisition, and data processing have significantly enhanced the applicability and performance of ECT. Here we describe a detailed workflow for an ECT experiment, including cell culture, vitrification, data acquisition, data reconstruction, tomogram analysis, and subtomogram averaging. This protocol provides an entry point to the technique for students and researchers and indicates the many possible variations arising from specific target properties and the available instrumentation. |
BibTeX:
@article{Weiss2017,
author = {Weiss, Gregor L and Medeiros, João M and Pilhofer, Martin},
title = {In Situ Imaging of Bacterial Secretion Systems by Electron Cryotomography.},
journal = {Methods in molecular biology (Clifton, N.J.)},
year = {2017},
volume = {1615},
pages = {353--375},
doi = {10.1007/978-1-4939-7033-9_27}
}
|
| West AM, Rosenberg SC, Ur SN, Lehmer MK, Ye Q, Hagemann G, Caballero I, Usón I, MacQueen AJ, Herzog F and Corbett KD (2019), "A conserved filamentous assembly underlies the structure of the meiotic chromosome axis.", eLife. Vol. 8 |
| Abstract: The meiotic chromosome axis plays key roles in meiotic chromosome organization and recombination, yet the underlying protein components of this structure are highly diverged. Here, we show that 'axis core proteins' from budding yeast (Red1), mammals (SYCP2/SYCP3), and plants (ASY3/ASY4) are evolutionarily related and play equivalent roles in chromosome axis assembly. We first identify 'closure motifs' in each complex that recruit meiotic HORMADs, the master regulators of meiotic recombination. We next find that axis core proteins form homotetrameric (Red1) or heterotetrameric (SYCP2:SYCP3 and ASY3:ASY4) coiled-coil assemblies that further oligomerize into micron-length filaments. Thus, the meiotic chromosome axis core in fungi, mammals, and plants shares a common molecular architecture, and likely also plays conserved roles in meiotic chromosome axis assembly and recombination control. |
BibTeX:
@article{West2019,
author = {West, Alan Mv and Rosenberg, Scott C and Ur, Sarah N and Lehmer, Madison K and Ye, Qiaozhen and Hagemann, Götz and Caballero, Iracema and Usón, Isabel and MacQueen, Amy J and Herzog, Franz and Corbett, Kevin D},
title = {A conserved filamentous assembly underlies the structure of the meiotic chromosome axis.},
journal = {eLife},
year = {2019},
volume = {8},
doi = {10.7554/eLife.40372}
}
|
| Wingfield JL, Mekonnen B, Mengoni I, Liu P, Jordan M, Diener D, Pigino G and Lechtreck K (2021), "In vivo imaging shows continued association of several IFT-A, IFT-B and dynein complexes while IFT trains U-turn at the tip.", Journal of cell science. Vol. 134 |
| Abstract: Flagellar assembly depends on intraflagellar transport (IFT), a bidirectional motility of protein carriers, the IFT trains. The trains are periodic assemblies of IFT-A and IFT-B subcomplexes and the motors kinesin-2 and IFT dynein. At the tip, anterograde trains are remodeled for retrograde IFT, a process that in Chlamydomonas involves kinesin-2 release and train fragmentation. However, the degree of train disassembly at the tip remains unknown. Here, we performed two-color imaging of fluorescent protein-tagged IFT components, which indicates that IFT-A and IFT-B proteins from a given anterograde train usually return in the same set of retrograde trains. Similarly, concurrent turnaround was typical for IFT-B proteins and the IFT dynein subunit D1bLIC-GFP but severance was observed as well. Our data support a simple model of IFT turnaround, in which IFT-A, IFT-B and IFT dynein typically remain associated at the tip and segments of the anterograde trains convert directly into retrograde trains. Continuous association of IFT-A, IFT-B and IFT dynein during tip remodeling could balance protein entry and exit, preventing the build-up of IFT material in flagella. |
BibTeX:
@article{Wingfield2021,
author = {Wingfield, Jenna L. and Mekonnen, Betlehem and Mengoni, Ilaria and Liu, Peiwei and Jordan, Mareike and Diener, Dennis and Pigino, Gaia and Lechtreck, Karl},
title = {In vivo imaging shows continued association of several IFT-A, IFT-B and dynein complexes while IFT trains U-turn at the tip.},
journal = {Journal of cell science},
year = {2021},
volume = {134},
doi = {10.1242/jcs.259010}
}
|
| Wolf SG and Elbaum M (2019), "CryoSTEM tomography in biology.", Methods in cell biology. Vol. 152, pp. 197-215. |
| Abstract: Electron cryo-tomography using the scanning transmission modality (STEM) enables 3D reconstruction of unstained, vitrified specimens as thick as 1μm or more. Contrast is related to mass/thickness and atomic number, providing quantifiable chemical characterization and mass mapping of intact prokaryotic and eukaryotic cells. Energy dispersive X-ray spectroscopy by STEM provides a simple, on-the-spot chemical identification of the elemental composition in sub-cellular organic bodies or mineral deposits. This chapter provides basic background and practical information for performing cryo-STEM tomography on vitrified biological cells. |
BibTeX:
@article{Wolf2019,
author = {Wolf, Sharon G and Elbaum, Michael},
title = {CryoSTEM tomography in biology.},
journal = {Methods in cell biology},
year = {2019},
volume = {152},
pages = {197--215},
doi = {10.1016/bs.mcb.2019.04.001}
}
|
| Woodward CL, Mendonça LM and Jensen GJ (2015), "Direct visualization of vaults within intact cells by electron cryo-tomography.", Cell Mol Life Sci. Vol. 72(17), pp. 3401-3409. |
| Abstract: The vault complex is the largest cellular ribonucleoprotein complex ever characterized and is present across diverse Eukarya. Despite significant information regarding the structure, composition and evolutionary conservation of the vault, little is know about the complex's actual biological function. To determine if intracellular vaults are morphologically similar to previously studied purified and recombinant vaults, we have used electron cryo-tomography to characterize the vault complexes found in the thin edges of primary human cells growing in tissue culture. Our studies confirm that intracellular vaults are similar in overall size and shape to purified and recombinant vaults previously analyzed. Results from subtomogram averaging indicate that densities within the vault lumen are not ordered, but randomly distributed. We also observe that vaults located in the extreme periphery of the cytoplasm predominately associate with granule-like structures and actin. Our ultrastructure studies augment existing biochemical, structural and genetic information on the vault, and provide important intracellular context for the ongoing efforts to understand the biological function of the native cytoplasmic vault. |
BibTeX:
@article{Woodward2015,
author = {Woodward, Cora L. and Mendonça, Luiza M. and Jensen, Grant J.},
title = {Direct visualization of vaults within intact cells by electron cryo-tomography.},
journal = {Cell Mol Life Sci},
school = {Division of Biology, California Institute of Technology, 1200 E California Blvd, Pasadena, CA, 91125, USA.},
year = {2015},
volume = {72},
number = {17},
pages = {3401--3409},
url = {http://dx.doi.org/10.1007/s00018-015-1898-y},
doi = {10.1007/s00018-015-1898-y}
}
|
| Xia X, Wu W, Cui Y, Roy P and Zhou ZH (2021), "Bluetongue virus capsid protein VP5 perforates membranes at low endosomal pH during viral entry.", Nature microbiology. Vol. 6, pp. 1424-1432. |
| Abstract: Bluetongue virus (BTV) is a non-enveloped virus and causes substantial morbidity and mortality in ruminants such as sheep. Fashioning a receptor-binding protein (VP2) and a membrane penetration protein (VP5) on the surface, BTV releases its genome-containing core (VP3 and VP7) into the host cell cytosol after perforation of the endosomal membrane. Unlike enveloped ones, the entry mechanisms of non-enveloped viruses into host cells remain poorly understood. Here we applied single-particle cryo-electron microscopy, cryo-electron tomography and structure-guided functional assays to characterize intermediate states of BTV cell entry in endosomes. Four structures of BTV at the resolution range of 3.4-3.9 Å show the different stages of structural rearrangement of capsid proteins on exposure to low pH, including conformational changes of VP5, stepwise detachment of VP2 and a small shift of VP7. In detail, sensing of the low-pH condition by the VP5 anchor domain triggers three major VP5 actions: projecting the hidden dagger domain, converting a surface loop to a protonated β-hairpin that anchors VP5 to the core and stepwise refolding of the unfurling domains into a six-helix stalk. Cryo-electron tomography structures of BTV interacting with liposomes show a length decrease of the VP5 stalk from 19.5 to 15.5 nm after its insertion into the membrane. Our structures, functional assays and structure-guided mutagenesis experiments combined indicate that this stalk, along with dagger domain and the WHXL motif, creates a single pore through the endosomal membrane that enables the viral core to enter the cytosol. Our study unveils the detailed mechanisms of BTV membrane penetration and showcases general methods to study cell entry of other non-enveloped viruses. |
BibTeX:
@article{Xia2021,
author = {Xia, Xian and Wu, Weining and Cui, Yanxiang and Roy, Polly and Zhou, Z. Hong},
title = {Bluetongue virus capsid protein VP5 perforates membranes at low endosomal pH during viral entry.},
journal = {Nature microbiology},
year = {2021},
volume = {6},
pages = {1424--1432},
doi = {10.1038/s41564-021-00988-8}
}
|
| Xiong Q, Morphew MK, Schwartz CL, Hoenger AH and Mastronarde DN (2009), "CTF determination and correction for low dose tomographic tilt series.", J Struct Biol. Vol. 168(3), pp. 378-387. |
| Abstract: The resolution of cryo-electron tomography can be limited by the first zero of the microscope's contrast transfer function (CTF). To achieve higher resolution, it is critical to determine the CTF and correct its phase inversions. However, the extremely low signal-to-noise ratio (SNR) and the defocus gradient in the projections of tilted specimens make this process challenging. Two programs, CTFPLOTTER and CTFPHASEFLIP, have been developed to address these issues. CTFPLOTTER obtains a 1D power spectrum by periodogram averaging and rotational averaging and it estimates the noise background with a novel approach, which uses images taken with no specimen. The background-subtracted 1D power spectra from image regions at different defocus values are then shifted to align their first zeros and averaged together. This averaging improves the SNR sufficiently that it becomes possible to determine the defocus for subsets of the tilt series rather than just the entire series. CTFPHASEFLIP corrects images line-by-line by inverting phases appropriately in thin strips of the image at nearly constant defocus. CTF correction by these methods is shown to improve the resolution of aligned, averaged particles extracted from tomograms. However, some restoration of Fourier amplitudes at high frequencies is important for seeing the benefits from CTF correction. |
BibTeX:
@article{Xiong2009,
author = {Xiong, Quanren and Morphew, Mary K. and Schwartz, Cindi L. and Hoenger, Andreas H. and Mastronarde, David N.},
title = {CTF determination and correction for low dose tomographic tilt series.},
journal = {J Struct Biol},
school = {Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309, USA.},
year = {2009},
volume = {168},
number = {3},
pages = {378--387},
url = {http://dx.doi.org/10.1016/j.jsb.2009.08.016},
doi = {10.1016/j.jsb.2009.08.016}
}
|
| Yamaguchi H, Oda T, Kikkawa M and Takeda H (2018), "Systematic studies of all PIH proteins in zebrafish reveal their distinct roles in axonemal dynein assembly.", eLife. Vol. 7 |
| Abstract: Construction of motile cilia/flagella requires cytoplasmic preassembly of axonemal dyneins before transport into cilia. Axonemal dyneins have various subtypes, but the roles of each dynein subtype and their assembly processes remain elusive in vertebrates. The PIH protein family, consisting of four members, has been implicated in the assembly of different dynein subtypes, although evidence for this idea is sparse. Here, we established zebrafish mutants of all four PIH-protein genes: , , , and , and analyzed the structures of axonemal dyneins in mutant spermatozoa by cryo-electron tomography. Mutations caused the loss of specific dynein subtypes, which was correlated with abnormal sperm motility. We also found organ-specific compositions of dynein subtypes, which could explain the severe motility defects of mutant Kupffer's vesicle cilia. Our data demonstrate that all vertebrate PIH proteins are differently required for cilia/flagella motions and the assembly of axonemal dyneins, assigning specific dynein subtypes to each PIH protein. |
BibTeX:
@article{Yamaguchi2018,
author = {Yamaguchi, Hiroshi and Oda, Toshiyuki and Kikkawa, Masahide and Takeda, Hiroyuki},
title = {Systematic studies of all PIH proteins in zebrafish reveal their distinct roles in axonemal dynein assembly.},
journal = {eLife},
year = {2018},
volume = {7},
doi = {10.7554/eLife.36979}
}
|
| Yang JE, Larson MR, Sibert BS, Kim JY, Parrell D, Sanchez JC, Pappas V, Kumar A, Cai K, Thompson K and Wright ER (), "Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies". |
BibTeX:
@misc{Yang,
author = {Jie E. Yang and Matthew R. Larson and Bryan S. Sibert and Joseph Y. Kim and Daniel Parrell and Juan C. Sanchez and Victoria Pappas and Anil Kumar and Kai Cai and Keith Thompson and Elizabeth R. Wright},
title = {Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies},
doi = {10.1101/2021.12.31.474669}
}
|
| Yang W and Briegel A (2018), "Use of Cryo-EM to Study the Structure of Chemoreceptor Arrays In Vivo.", Methods in molecular biology (Clifton, N.J.). Vol. 1729, pp. 173-185. |
| Abstract: Cryo-electron microscopy (cryo-EM) allows the imaging of intact macromolecular complexes in the context of whole cells. The biological samples for cryo-EM are kept in a near-native state by flash freezing, without the need for any additional sample preparation or fixation steps. Since transmission electron microscopy only generates 2D projections of the samples, the specimen has to be tilted in order to recover its 3D structural information. This is done by collecting images of the sample with various tilt angles in respect to the electron beam. The acquired tilt series can then be computationally back-projected. This technique is called electron cryotomography (ECT), and has been instrumental in unraveling the architecture of chemoreceptor arrays. Here we describe the method of visualizing in vivo bacterial chemoreceptor arrays in three main steps: immobilization of bacterial cells on EM grids by plunge-freezing; 2D image acquisition in tilt series; and 3D tomogram reconstruction. |
BibTeX:
@article{Yang2018a,
author = {Yang, Wen and Briegel, Ariane},
title = {Use of Cryo-EM to Study the Structure of Chemoreceptor Arrays In Vivo.},
journal = {Methods in molecular biology (Clifton, N.J.)},
year = {2018},
volume = {1729},
pages = {173--185},
doi = {10.1007/978-1-4939-7577-8_16}
}
|
| Zabeo D, Croft JT and Höög JL (2019), "Axonemal doublet microtubules can split into two complete singlets in human sperm flagellum tips.", FEBS letters. |
| Abstract: Motile flagella are crucial for human fertility and embryonic development. The distal tip of the flagellum is where growth and intra-flagellar transport are coordinated. In most model organisms, but not all, the distal tip includes a 'singlet region', where axonemal doublet microtubules (dMT) terminate and only complete A-tubules extend as singlet microtubules (sMT) to the tip. How a human flagellar tip is structured is unknown. Here, the flagellar tip structure of human spermatozoa was investigated by cryo-electron tomography, revealing the formation of a complete sMT from both the A-tubule and B-tubule of dMTs. This different tip arrangement in human spermatozoa shows the need to investigate human flagella directly in order to understand their role in health and disease. |
BibTeX:
@article{Zabeo2019,
author = {Zabeo, Davide and Croft, Jacob T and Höög, Johanna L},
title = {Axonemal doublet microtubules can split into two complete singlets in human sperm flagellum tips.},
journal = {FEBS letters},
year = {2019},
doi = {10.1002/1873-3468.13379}
}
|
| Zabeo D, Heumann JM, Schwartz CL, Suzuki-Shinjo A, Morgan G, Widlund PO and Höög JL (2018), "A lumenal interrupted helix in human sperm tail microtubules.", Scientific reports. Vol. 8, pp. 2727. |
| Abstract: Eukaryotic flagella are complex cellular extensions involved in many human diseases gathered under the term ciliopathies. Currently, detailed insights on flagellar structure come mostly from studies on protists. Here, cryo-electron tomography (cryo-ET) was performed on intact human spermatozoon tails and showed a variable number of microtubules in the singlet region (inside the end-piece). Inside the microtubule plus end, a novel left-handed interrupted helix which extends several micrometers was discovered. This structure was named Tail Axoneme Intra-Lumenal Spiral (TAILS) and binds directly to 11 protofilaments on the internal microtubule wall, in a coaxial fashion with the surrounding microtubule lattice. It leaves a gap over the microtubule seam, which was directly visualized in both singlet and doublet microtubules. We speculate that TAILS may stabilize microtubules, enable rapid swimming or play a role in controlling the swimming direction of spermatozoa. |
BibTeX:
@article{Zabeo2018,
author = {Zabeo, Davide and Heumann, John M and Schwartz, Cindi L and Suzuki-Shinjo, Azusa and Morgan, Garry and Widlund, Per O and Höög, Johanna L},
title = {A lumenal interrupted helix in human sperm tail microtubules.},
journal = {Scientific reports},
year = {2018},
volume = {8},
pages = {2727},
doi = {10.1038/s41598-018-21165-8}
}
|
| Zaman R, Vernekhol D, Azubel M, Kornberg R and Xing L (2018), "Distinctive Energy Profile of Water-Soluble, Thiolate-Protected Gold Nanoparticles as Potential Molecular Marker for XECTImaging", In MEDICAL PHYSICS. Vol. 45(6), pp. E538-E538.
[BibTeX] |
BibTeX:
@inproceedings{Zaman2018,
author = {Zaman, R and Vernekhol, D and Azubel, M and Kornberg, R and Xing, L},
title = {Distinctive Energy Profile of Water-Soluble, Thiolate-Protected Gold Nanoparticles as Potential Molecular Marker for XECTImaging},
booktitle = {MEDICAL PHYSICS},
year = {2018},
volume = {45},
number = {6},
pages = {E538--E538}
}
|
| Zeev-Ben-Mordehai T, Vasishtan D, Hernández Durán A, Vollmer B, White P, Prasad Pandurangan A, Siebert CA, Topf M and Grünewald K (2016), "Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B.", Proc Natl Acad Sci U S A. Vol. 113(15), pp. 4176-4181. |
| Abstract: Many viruses are enveloped by a lipid bilayer acquired during assembly, which is typically studded with one or two types of glycoproteins. These viral surface proteins act as the primary interface between the virus and the host. Entry of enveloped viruses relies on specialized fusogen proteins to help merge the virus membrane with the host membrane. In the multicomponent herpesvirus fusion machinery, glycoprotein B (gB) acts as this fusogen. Although the structure of the gB ectodomain postfusion conformation has been determined, any other conformations (e.g., prefusion, intermediate conformations) have so far remained elusive, thus restricting efforts to develop antiviral treatments and prophylactic vaccines. Here, we have characterized the full-length herpes simplex virus 1 gB in a native membrane by displaying it on cell-derived vesicles and using electron cryotomography. Alongside the known postfusion conformation, a novel one was identified. Its structure, in the context of the membrane, was determined by subvolume averaging and found to be trimeric like the postfusion conformation, but appeared more condensed. Hierarchical constrained density-fitting of domains unexpectedly revealed the fusion loops in this conformation to be apart and pointing away from the anchoring membrane. This vital observation is a substantial step forward in understanding the complex herpesvirus fusion mechanism, and opens up new opportunities for more targeted intervention of herpesvirus entry. |
BibTeX:
@article{Zeev-Ben-Mordehai2016,
author = {Zeev-Ben-Mordehai, Tzviya and Vasishtan, Daven and Hernández Durán, Anna and Vollmer, Benjamin and White, Paul and Prasad Pandurangan, Arun and Siebert, C Alistair and Topf, Maya and Grünewald, Kay},
title = {Two distinct trimeric conformations of natively membrane-anchored full-length herpes simplex virus 1 glycoprotein B.},
journal = {Proc Natl Acad Sci U S A},
school = {Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, United Kingdom; kay@strubi.ox.ac.uk.},
year = {2016},
volume = {113},
number = {15},
pages = {4176--4181},
url = {http://dx.doi.org/10.1073/pnas.1523234113},
doi = {10.1073/pnas.1523234113}
}
|
| Zeev-Ben-Mordehai T, Vasishtan D, Siebert CA and Grünewald K (2014), "The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane.", Nat Commun. Vol. 5, pp. 3912. |
| Abstract: Fusogens are membrane proteins that remodel lipid bilayers to facilitate membrane merging. Although several fusogen ectodomain structures have been solved, structural information on full-length, natively membrane-anchored fusogens is scarce. Here we present the electron cryo microscopy three-dimensional reconstruction of the Caenorhabditis elegans epithelial fusion failure 1 (EFF-1) protein natively anchored in cell-derived membrane vesicles. This reveals a membrane protruding, asymmetric, elongated monomer. Flexible fitting of a protomer of the EFF-1 crystal structure, which is homologous to viral class-II fusion proteins, shows that EFF-1 has a hairpin monomeric conformation before fusion. These structural insights, when combined with our observations of membrane-merging intermediates between vesicles, enable us to propose a model for EFF-1 mediated fusion. This process, involving identical proteins on both membranes to be fused, follows a mechanism that shares features of SNARE-mediated fusion while using the structural building blocks of the unilaterally acting class-II viral fusion proteins. |
BibTeX:
@article{Zeev-Ben-Mordehai2014a,
author = {Zeev-Ben-Mordehai, Tzviya and Vasishtan, Daven and Siebert, C Alistair and Grünewald, Kay},
title = {The full-length cell-cell fusogen EFF-1 is monomeric and upright on the membrane.},
journal = {Nat Commun},
school = {Oxford Particle Imaging Centre, Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK.},
year = {2014},
volume = {5},
pages = {3912},
url = {http://dx.doi.org/10.1038/ncomms4912},
doi = {10.1038/ncomms4912}
}
|
| Zeev-Ben-Mordehai T, Vasishtan D, Siebert CA, Whittle C and Grünewald K (2014), "Extracellular vesicles: a platform for the structure determination of membrane proteins by Cryo-EM.", Structure. Vol. 22(11), pp. 1687-1692. |
| Abstract: Membrane protein-enriched extracellular vesicles (MPEEVs) provide a platform for studying intact membrane proteins natively anchored with the correct topology in genuine biological membranes. This approach circumvents the need to conduct tedious detergent screens for solubilization, purification, and reconstitution required in classical membrane protein studies. We have applied this method to three integral type I membrane proteins, namely the Caenorhabditis elegans cell-cell fusion proteins AFF-1 and EFF-1 and the glycoprotein B (gB) from Herpes simplex virus type 1 (HSV1). Electron cryotomography followed by subvolume averaging allowed the 3D reconstruction of EFF-1 and HSV1 gB in the membrane as well as an analysis of the spatial distribution and interprotein interactions on the membrane. MPEEVs have many applications beyond structural/functional investigations, such as facilitating the raising of antibodies, for protein-protein interaction assays or for diagnostics use, as biomarkers, and possibly therapeutics. |
BibTeX:
@article{Zeev-Ben-Mordehai2014,
author = {Zeev-Ben-Mordehai, Tzviya and Vasishtan, Daven and Siebert, C Alistair and Whittle, Cathy and Grünewald, Kay},
title = {Extracellular vesicles: a platform for the structure determination of membrane proteins by Cryo-EM.},
journal = {Structure},
school = {Division of Structural Biology, Wellcome Trust Centre for Human Genetics, University of Oxford, Oxford OX3 7BN, UK. Electronic address: kay@strubi.ox.ac.uk.},
year = {2014},
volume = {22},
number = {11},
pages = {1687--1692},
url = {http://dx.doi.org/10.1016/j.str.2014.09.005},
doi = {10.1016/j.str.2014.09.005}
}
|
| Zettl T, Das R, Harbury PA, Herschlag D, Lipfert J, Mathew RS and Shi X (2018), "Recording and Analyzing Nucleic Acid Distance Distributions with X-Ray Scattering Interferometry (XSI)", Current protocols in nucleic acid chemistry. Vol. 73(1), pp. e54. Wiley Online Library.
[BibTeX] |
BibTeX:
@article{Zettl2018,
author = {Zettl, Thomas and Das, Rhiju and Harbury, Pehr AB and Herschlag, Daniel and Lipfert, Jan and Mathew, Rebecca S and Shi, Xuesong},
title = {Recording and Analyzing Nucleic Acid Distance Distributions with X-Ray Scattering Interferometry (XSI)},
journal = {Current protocols in nucleic acid chemistry},
publisher = {Wiley Online Library},
year = {2018},
volume = {73},
number = {1},
pages = {e54}
}
|
| Zhang J, Wang H, Imhof S, Zhou X, Liao S, Atanasov I, Hui WH, Hill KL and Zhou ZH (2021), "Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells.", Cell discovery. Vol. 7, pp. 51. |
| Abstract: Eukaryotic flagella (synonymous with cilia) rely on a microtubule-based axoneme, together with accessory filaments to carryout motility and signaling functions. While axoneme structures are well characterized, 3D ultrastructure of accessory filaments and their axoneme interface are mostly unknown, presenting a critical gap in understanding structural foundations of eukaryotic flagella. In the flagellum of the protozoan parasite Trypanosoma brucei (T. brucei), the axoneme is accompanied by a paraflagellar rod (PFR) that supports non-planar motility and signaling necessary for disease transmission and pathogenesis. Here, we employed cryogenic electron tomography (cryoET) with sub-tomographic averaging, to obtain structures of the PFR, PFR-axoneme connectors (PACs), and the axonemal central pair complex (CPC). The structures resolve how the 8 nm repeat of the axonemal tubulin dimer interfaces with the 54 nm repeat of the PFR, which consist of proximal, intermediate, and distal zones. In the distal zone, stacked "density scissors" connect with one another to form a "scissors stack network (SSN)" plane oriented 45° to the axoneme axis; and 370 parallel SSN planes are connected by helix-rich wires into a paracrystalline array with 90% empty space. Connections from these wires to the intermediate zone, then to overlapping layers of the proximal zone and to the PACs, and ultimately to the CPC, point to a contiguous pathway for signal transmission. Together, our findings provide insights into flagellum-driven, non-planar helical motility of T. brucei and have broad implications ranging from cell motility and tensegrity in biology, to engineering principles in bionics. |
BibTeX:
@article{Zhang2021,
author = {Zhang, Jiayan and Wang, Hui and Imhof, Simon and Zhou, Xueting and Liao, Shiqing and Atanasov, Ivo and Hui, Wong H. and Hill, Kent L. and Zhou, Z. Hong},
title = {Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells.},
journal = {Cell discovery},
year = {2021},
volume = {7},
pages = {51},
doi = {10.1038/s41421-021-00281-2}
}
|
| Zhang J, Wang H, Imhof S, Zhou X, Liao S, Atanasov I, Hui WH, Hill KL and Zhou ZH (2021), "Author Correction: Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells.", Cell discovery. Vol. 7, pp. 61. |
BibTeX:
@article{Zhang2021a,
author = {Zhang, Jiayan and Wang, Hui and Imhof, Simon and Zhou, Xueting and Liao, Shiqing and Atanasov, Ivo and Hui, Wong H. and Hill, Kent L. and Zhou, Z. Hong},
title = {Author Correction: Structure of the trypanosome paraflagellar rod and insights into non-planar motility of eukaryotic cells.},
journal = {Cell discovery},
year = {2021},
volume = {7},
pages = {61},
doi = {10.1038/s41421-021-00317-7}
}
|
| Zhao X, Schwartz CL, Pierson J, Giovannoni SJ, McIntosh JR and Nicastro D (2017), "Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium "Candidatus Pelagibacter ubique".", Applied and environmental microbiology. Vol. 83 |
| Abstract: SAR11 bacteria are small, heterotrophic, marine alphaproteobacteria found throughout the oceans. They thrive at the low nutrient concentrations typical of open ocean conditions, although the adaptations required for life under those conditions are not well understood. To illuminate this issue, we used cryo-electron tomography to study "Candidatus Pelagibacter ubique" strain HTCC1062, a member of the SAR11 clade. Our results revealed its cellular dimensions and details of its intracellular organization. Frozen-hydrated cells, which were preserved in a life-like state, had an average cell volume (enclosed by the outer membrane) of 0.037 ± 0.011 μm(3) Strikingly, the periplasmic space occupied ∼20% to 50% of the total cell volume in log-phase cells and ∼50% to 70% in stationary-phase cells. The nucleoid occupied the convex side of the crescent-shaped cells and the ribosomes predominantly occupied the concave side, at a relatively high concentration of 10,000 to 12,000 ribosomes/μm(3) Outer membrane pore complexes, likely composed of PilQ, were frequently observed in both log-phase and stationary-phase cells. Long filaments, most likely type IV pili, were found on dividing cells. The physical dimensions, intracellular organization, and morphological changes throughout the life cycle of "Ca. Pelagibacter ubique" provide structural insights into the functional adaptions of these oligotrophic ultramicrobacteria to their habitat. Bacterioplankton of the SAR11 clade (Pelagibacterales) are of interest because of their global biogeochemical significance and because they appear to have been molded by unusual evolutionary circumstances that favor simplicity and efficiency. They have adapted to an ecosystem in which nutrient concentrations are near the extreme limits at which transport systems can function adequately, and they have evolved streamlined genomes to execute only functions essential for life. However, little is known about the actual size limitations and cellular features of living oligotrophic ultramicrobacteria. In this study, we have used cryo-electron tomography to obtain accurate physical information about the cellular architecture of "Candidatus Pelagibacter ubique," the first cultivated member of the SAR11 clade. These results provide foundational information for answering questions about the cell architecture and functions of these ultrasmall oligotrophic bacteria. |
BibTeX:
@article{Zhao2017,
author = {Zhao, Xiaowei and Schwartz, Cindi L and Pierson, Jason and Giovannoni, Stephen J and McIntosh, J Richard and Nicastro, Daniela},
title = {Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium "Candidatus Pelagibacter ubique".},
journal = {Applied and environmental microbiology},
year = {2017},
volume = {83},
doi = {10.1128/AEM.02807-16}
}
|
| Zhao Y, Pinskey J, Lin J, Yin W, Sears PR, Daniels LA, Zariwala MA, Knowles MR, Ostrowski LE and Nicastro D (2021), "Structural insights into the cause of human , javax.xml.bind.JAXBElement@61eb468d, primary ciliary dyskinesia.", Molecular biology of the cell. Vol. 32, pp. 1202-1209. |
| Abstract: Cilia and flagella are evolutionarily conserved eukaryotic organelles involved in cell motility and signaling. In humans, mutations in Radial Spoke Head Component 4A ( ) can lead to primary ciliary dyskinesia (PCD), a life-shortening disease characterized by chronic respiratory tract infections, abnormal organ positioning, and infertility. Despite its importance for human health, the location of RSPH4A in human cilia has not been resolved, and the structural basis of PCD remains elusive. Here, we present the native three-dimensional structure of human respiratory cilia using samples collected noninvasively from a PCD patient. Using cryo-electron tomography (cryo-ET) and subtomogram averaging, we compared the structures of control and cilia, revealing primary defects in two of the three radial spokes (RSs) within the axonemal repeat and secondary (heterogeneous) defects in the central pair complex. Similar to cilia, the radial spoke heads of RS1 and RS2, but not RS3, were missing in cilia. However, cilia also exhibited defects within the arch domains adjacent to the RS1 and RS2 heads, which were not observed with RSPH1 loss. Our results provide insight into the underlying structural basis for PCD and highlight the benefits of applying cryo-ET directly to patient samples for molecular structure determination. |
BibTeX:
@article{Zhao2021,
author = {Zhao, Yanhe and Pinskey, Justine and Lin, Jianfeng and Yin, Weining and Sears, Patrick R. and Daniels, Leigh A. and Zariwala, Maimoona A. and Knowles, Michael R. and Ostrowski, Lawrence E. and Nicastro, Daniela},
title = {Structural insights into the cause of human , javax.xml.bind.JAXBElement@61eb468d, primary ciliary dyskinesia.},
journal = {Molecular biology of the cell},
year = {2021},
volume = {32},
pages = {1202--1209},
doi = {10.1091/mbc.E20-12-0806}
}
|
| Fu G, Augspurger K, Sakizadeh J, Reck J, Bower R, Tritschler D, Gui L, Nicastro D and Porter ME (2023), "The ciliary MBO2 complex targets assembly of inner arm dynein b and reveals additional doublet microtubule asymmetries.", bioRxiv : the preprint server for biology. |
| Abstract: Ciliary motility requires the spatiotemporal coordination of multiple dynein motors by regulatory complexes located within the 96 nm axoneme repeat. Many organisms can alter ciliary waveforms in response to internal or external stimuli, but little is known about the specific polypeptides and structural organization of complexes that regulate waveforms. In Chlamydomonas, several mutations convert the ciliary waveform from an asymmetric, ciliary-type stroke to a symmetric, flagellar-type stroke. Some of these mutations alter subunits located at the inner junction of the doublet microtubule and others alter interactions between the dynein arms and the radial spokes. These and other axonemal substructures are interconnected by a network of poorly characterized proteins. Here we re-analyze several motility mutants (mbo, fap57, pf12/pacrg) to identify new components in this network. The mbo (move backwards only) mutants are unable to swim forwards with an asymmetric waveform. Proteomics identified more than 19 polypeptides that are missing or reduced in mbo mutants, including one inner dynein arm, IDA b. Several MBO2-associated proteins are also altered in fap57 and pf12/parcg mutants, suggesting overlapping networks. Two subunits are highly conserved, coiled coil proteins found in other species with motile cilia and others contain potential signaling domains. Cryo-electron tomography and epitope tagging revealed that the MBO2 complex is found on specific doublet microtubules and forms a large, L-shaped structure that contacts the base of IDA b that interconnects multiple dynein regulatory complexes and varies in a doublet microtubule specific fashion. |
BibTeX:
@article{Fu2023,
author = {Fu, Gang and Augspurger, Katherine and Sakizadeh, Jason and Reck, Jaimee and Bower, Raqual and Tritschler, Douglas and Gui, Long and Nicastro, Daniela and Porter, Mary E.},
title = {The ciliary MBO2 complex targets assembly of inner arm dynein b and reveals additional doublet microtubule asymmetries.},
journal = {bioRxiv : the preprint server for biology},
year = {2023},
doi = {10.1101/2023.07.31.551375}
}
|
| Gui L, O'Shaughnessy WJ, Cai K, Reetz E, Reese ML and Nicastro D (2023), "Cryo-tomography reveals rigid-body motion and organization of apicomplexan invasion machinery.", Nature communications. Vol. 14, pp. 1775. |
| Abstract: The apical complex is a specialized collection of cytoskeletal and secretory machinery in apicomplexan parasites, which include the pathogens that cause malaria and toxoplasmosis. Its structure and mechanism of motion are poorly understood. We used cryo-FIB-milling and cryo-electron tomography to visualize the 3D-structure of the apical complex in its protruded and retracted states. Averages of conoid-fibers revealed their polarity and unusual nine-protofilament arrangement with associated proteins connecting and likely stabilizing the fibers. Neither the structure of the conoid-fibers nor the architecture of the spiral-shaped conoid complex change during protrusion or retraction. Thus, the conoid moves as a rigid body, and is not spring-like and compressible, as previously suggested. Instead, the apical-polar-rings (APR), previously considered rigid, dilate during conoid protrusion. We identified actin-like filaments connecting the conoid and APR during protrusion, suggesting a role during conoid movements. Furthermore, our data capture the parasites in the act of secretion during conoid protrusion. |
BibTeX:
@article{Gui2023,
author = {Gui, Long and O'Shaughnessy, William J. and Cai, Kai and Reetz, Evan and Reese, Michael L. and Nicastro, Daniela},
title = {Cryo-tomography reveals rigid-body motion and organization of apicomplexan invasion machinery.},
journal = {Nature communications},
year = {2023},
volume = {14},
pages = {1775},
doi = {10.1038/s41467-023-37327-w}
}
|
| Bousquet C, Heumann JM, Chrétien D and Guyomar C (2023), "Characterization of Microtubule Lattice Heterogeneity by Segmented Subtomogram Averaging.", Bio-protocol. Vol. 13, pp. e4723. |
| Abstract: Microtubule structure is commonly investigated using single-particle analysis (SPA) or subtomogram averaging (STA), whose main objectives are to gather high-resolution information on the αβ-tubulin heterodimer and on its interactions with neighboring molecules within the microtubule lattice. The maps derived from SPA approaches usually delineate a continuous organization of the αβ-tubulin heterodimer that alternate regularly head-to-tail along protofilaments, and that share homotypic lateral interactions between monomers (α-α, β-β), except at one unique region called the seam, made of heterotypic ones (α-β, β-α). However, this textbook description of the microtubule lattice has been challenged over the years by several studies that revealed the presence of multi-seams in microtubules assembled in vitro from purified tubulin. To analyze in deeper detail their intrinsic structural heterogeneity, we have developed a segmented subtomogram averaging (SSTA) strategy on microtubules decorated with kinesin motor-domains that bind every αβ-tubulin heterodimer. Individual protofilaments and microtubule centers are modeled, and sub-volumes are extracted at every kinesin motor domain position to obtain full subtomogram averages of the microtubules. The model is divided into shorter segments, and subtomogram averages of each segment are calculated using the main parameters of the full-length microtubule settings as a template. This approach reveals changes in the number and location of seams within individual microtubules assembled in vitro from purified tubulin and in Xenopus egg cytoplasmic extracts. Key features This protocol builds upon the method developed by J.M. Heumann to perform subtomogram averages of microtubules and extends it to divide them into shorter segments. Microtubules are decorated with kinesin motor-domains to determine the underlying organization of its constituent αβ-tubulin heterodimers. The SSTA approach allows analysis of the structural heterogeneity of individual microtubules and reveals multi-seams and changes in their number and location within their shaft. Graphical overview. |
BibTeX:
@article{Bousquet2023,
author = {Bousquet, Clément and Heumann, John M. and Chrétien, Denis and Guyomar, Charlotte},
title = {Characterization of Microtubule Lattice Heterogeneity by Segmented Subtomogram Averaging.},
journal = {Bio-protocol},
year = {2023},
volume = {13},
pages = {e4723},
doi = {10.21769/BioProtoc.4723}
}
|
| Yamaguchi H, Morikawa M and Kikkawa M (2023), "Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates.", eLife. Vol. 12 |
| Abstract: Outer arm dynein (OAD) is the main force generator of ciliary beating. Although OAD loss is the most frequent cause of human primary ciliary dyskinesia, the docking mechanism of OAD onto the ciliary doublet microtubule (DMT) remains elusive in vertebrates. Here, we analyzed the functions of Calaxin/Efcab1 and Armc4, the two of five components of vertebrate OAD-DC (docking complex), using zebrafish spermatozoa and cryo-electron tomography. Mutation of armc4 caused complete loss of OAD, whereas mutation of calaxin caused only partial loss of OAD. Detailed structural analysis revealed that calaxin (-/-)OADs are tethered to DMT through DC components other than Calaxin, and that recombinant Calaxin can autonomously rescue the deficient DC structure and the OAD instability. Our data demonstrate the discrete roles of Calaxin and Armc4 in the OAD-DMT interaction, suggesting the stabilizing process of OAD docking onto DMT in vertebrates. |
BibTeX:
@article{Yamaguchi2023,
author = {Yamaguchi, Hiroshi and Morikawa, Motohiro and Kikkawa, Masahide},
title = {Calaxin stabilizes the docking of outer arm dyneins onto ciliary doublet microtubule in vertebrates.},
journal = {eLife},
year = {2023},
volume = {12},
doi = {10.7554/eLife.84860}
}
|
| Oda T, Yanagisawa H, Kikkawa M and Kita Y (2023), "Unveiling Liquid-Crystalline Lipids in the Urothelial Membrane through Cryo-EM.", Research square. |
| Abstract: The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this investigation, we employed cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex embedded within the porcine AUM at a resolution of 3.5 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane. |
BibTeX:
@article{Oda2023,
author = {Oda, Toshiyuki and Yanagisawa, Haruaki and Kikkawa, Masahide and Kita, Yoshihiro},
title = {Unveiling Liquid-Crystalline Lipids in the Urothelial Membrane through Cryo-EM.},
journal = {Research square},
year = {2023},
doi = {10.21203/rs.3.rs-3080731/v1}
}
|
| Wang H, Zhang J, Toso D, Liao S, Sedighian F, Gunsalus R and Zhou ZH (2023), "Hierarchical organization and assembly of the archaeal cell sheath from an amyloid-like protein.", Nature communications. Vol. 14, pp. 6720. |
| Abstract: Certain archaeal cells possess external proteinaceous sheath, whose structure and organization are both unknown. By cellular cryogenic electron tomography (cryoET), here we have determined sheath organization of the prototypical archaeon, Methanospirillum hungatei. Fitting of Alphafold-predicted model of the sheath protein (SH) monomer into the 7.9 Å-resolution structure reveals that the sheath cylinder consists of axially stacked β-hoops, each of which is comprised of two to six 400 nm-diameter rings of β-strand arches (β-rings). With both similarities to and differences from amyloid cross-β fibril architecture, each β-ring contains two giant β-sheets contributed by 450 SH monomers that entirely encircle the outer circumference of the cell. Tomograms of immature cells suggest models of sheath biogenesis: oligomerization of SH monomers into β-ring precursors after their membrane-proximal cytoplasmic synthesis, followed by translocation through the unplugged end of a dividing cell, and insertion of nascent β-hoops into the immature sheath cylinder at the junction of two daughter cells. |
BibTeX:
@article{Wang2023,
author = {Wang, Hui and Zhang, Jiayan and Toso, Daniel and Liao, Shiqing and Sedighian, Farzaneh and Gunsalus, Robert and Zhou, Z. Hong},
title = {Hierarchical organization and assembly of the archaeal cell sheath from an amyloid-like protein.},
journal = {Nature communications},
year = {2023},
volume = {14},
pages = {6720},
doi = {10.1038/s41467-023-42368-2}
}
|
| Hwang JY, Chai P, Nawaz S, Choi J, Lopez-Giraldez F, Hussain S, Bilguvar K, Mane S, Lifton RP, Ahmad W, Zhang K and Chung J-J (2023), "LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility.", eLife. Vol. 12 |
| Abstract: Radial spokes (RS) are T-shaped multiprotein complexes on the axonemal microtubules. Repeated RS1, RS2, and RS3 couple the central pair to modulate ciliary and flagellar motility. Despite the cell type specificity of RS3 substructures, their molecular components remain largely unknown. Here, we report that a leucine-rich repeat-containing protein, LRRC23, is an RS3 head component essential for its head assembly and flagellar motility in mammalian spermatozoa. From infertile male patients with defective sperm motility, we identified a splice site variant of LRRC23 . A mutant mouse model mimicking this variant produces a truncated LRRC23 at the C-terminus that fails to localize to the sperm tail, causing male infertility due to defective sperm motility. LRRC23 was previously proposed to be an ortholog of the RS stalk protein RSP15. However, we found that purified recombinant LRRC23 interacts with an RS head protein RSPH9, which is abolished by the C-terminal truncation. Evolutionary and structural comparison also shows that LRRC34, not LRRC23, is the RSP15 ortholog. Cryo-electron tomography clearly revealed that the absence of the RS3 head and the sperm-specific RS2-RS3 bridge structure in LRRC23 mutant spermatozoa. Our study provides new insights into the structure and function of RS3 in mammalian spermatozoa and the molecular pathogenicity of LRRC23 underlying reduced sperm motility in infertile human males. |
BibTeX:
@article{Hwang2023,
author = {Hwang, Jae Yeon and Chai, Pengxin and Nawaz, Shoaib and Choi, Jungmin and Lopez-Giraldez, Francesc and Hussain, Shabir and Bilguvar, Kaya and Mane, Shrikant and Lifton, Richard P. and Ahmad, Wasim and Zhang, Kai and Chung, Jean-Ju},
title = {LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility.},
journal = {eLife},
year = {2023},
volume = {12},
doi = {10.7554/eLife.90095}
}
|
| Shimogawa MM, Wijono AS, Wang H, Zhang J, Sha J, Szombathy N, Vadakkan S, Pelayo P, Jonnalagadda K, Wohlschlegel J, Zhou ZH and Hill KL (2023), "FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction.", Nature communications. Vol. 14, pp. 5225. |
| Abstract: Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability and motility and provide lineage-specific adaptations, but individual MIP functions and assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei, that FAP106, a conserved MIP at the DMT inner junction, is required for trypanosome motility and functions as a critical interaction hub, directing assembly of several conserved and lineage-specific MIPs. We use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify MIP candidates. Using RNAi knockdown together with fitting of AlphaFold models into cryoET maps, we demonstrate that one of these candidates, MC8, is a trypanosome-specific MIP required for parasite motility. Our work advances understanding of MIP assembly mechanisms and identifies lineage-specific motility proteins that are attractive targets to consider for therapeutic intervention. |
BibTeX:
@article{Shimogawa2023,
author = {Shimogawa, Michelle M. and Wijono, Angeline S. and Wang, Hui and Zhang, Jiayan and Sha, Jihui and Szombathy, Natasha and Vadakkan, Sabeeca and Pelayo, Paula and Jonnalagadda, Keya and Wohlschlegel, James and Zhou, Z. Hong and Hill, Kent L.},
title = {FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction.},
journal = {Nature communications},
year = {2023},
volume = {14},
pages = {5225},
doi = {10.1038/s41467-023-40230-z}
}
|
| Zoratto S, Heuser T, Friedbacher G, Pletzenauer R, Graninger M, Marchetti-Deschmann M and Weiss VU (2023), "Adeno-Associated Virus-like Particles' Response to pH Changes as Revealed by nES-DMA.", Viruses. Vol. 15 |
| Abstract: Gas-phase electrophoresis on a nano-Electrospray Gas-phase Electrophoretic Mobility Molecular Analyzer (nES GEMMA) separates single-charged, native analytes according to the surface-dry particle size. A volatile electrolyte, often ammonium acetate, is a prerequisite for electrospraying. Over the years, nES GEMMA has demonstrated its unique capability to investigate (bio-)nanoparticle containing samples in respect to composition, analyte size, size distribution, and particle numbers. Virus-like particles (VLPs), being non-infectious vectors, are often employed for gene therapy applications. Focusing on adeno-associated virus 8 (AAV8) based VLPs, we investigated the response of these bionanoparticles to pH changes via nES GEMMA as ammonium acetate is known to exhibit these changes upon electrospraying. Indeed, slight yet significant differences in VLP diameters in relation to pH changes are found between empty and DNA-cargo-filled assemblies. Additionally, filled VLPs exhibit aggregation in dependence on the applied electrolyte's pH, as corroborated by atomic force microscopy. In contrast, cryogenic transmission electron microscopy did not relate to changes in the overall particle size but in the substantial particle's shape based on cargo conditions. Overall, we conclude that for VLP characterization, the pH of the applied electrolyte solution has to be closely monitored, as variations in pH might account for drastic changes in particles and VLP behavior. Likewise, extrapolation of VLP behavior from empty to filled particles has to be carried out with caution. |
BibTeX:
@article{Zoratto2023,
author = {Zoratto, Samuele and Heuser, Thomas and Friedbacher, Gernot and Pletzenauer, Robert and Graninger, Michael and Marchetti-Deschmann, Martina and Weiss, Victor U.},
title = {Adeno-Associated Virus-like Particles' Response to pH Changes as Revealed by nES-DMA.},
journal = {Viruses},
year = {2023},
volume = {15},
doi = {10.3390/v15061361}
}
|
| Chen JK, Liu T, Cai S, Ruan W, Ng CT, Shi J, Surana U and Gan L (2023), "Nanoscale analysis of human G1 and metaphase chromatin in situ" Cold Spring Harbor Laboratory. |
BibTeX:
@article{Chen2023,
author = {Chen, Jon Ken and Liu, Tingsheng and Cai, Shujun and Ruan, Weimei and Ng, Cai Tong and Shi, Jian and Surana, Uttam and Gan, Lu},
title = {Nanoscale analysis of human G1 and metaphase chromatin in situ},
publisher = {Cold Spring Harbor Laboratory},
year = {2023},
doi = {10.1101/2023.07.31.551204}
}
|
| Viar GA, Klena N, Martino F, Nievergelt A and Pigino G (2023), "The Tubulin Nano-Code: a protofilament-specific pattern of tubulin post-translational modifications regulates ciliary beating mechanics" Cold Spring Harbor Laboratory. |
BibTeX:
@article{Viar2023,
author = {Viar, G. Alvarez and Klena, N. and Martino, F. and Nievergelt, A. and Pigino, G.},
title = {The Tubulin Nano-Code: a protofilament-specific pattern of tubulin post-translational modifications regulates ciliary beating mechanics},
publisher = {Cold Spring Harbor Laboratory},
year = {2023},
doi = {10.1101/2023.06.28.546853}
}
|
| Yang JE, Larson MR, Sibert BS, Kim JY, Parrell D, Sanchez JC, Pappas V, Kumar A, Cai K, Thompson K and Wright ER (2023), "Correlative montage parallel array cryo-tomography for in situ structural cell biology.", Nature methods. Vol. 20, pp. 1537-1543. |
| Abstract: Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here we present robust tools for montage parallel array cryo-tomography (MPACT) tailored for vitrified specimens. The combination of correlative cryo-fluorescence microscopy, focused-ion-beam milling, substrate micropatterning, and MPACT supports studies that contextually define the three-dimensional architecture of cells. To further extend the flexibility of MPACT, tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis. |
BibTeX:
@article{Yang2023,
author = {Yang, Jie E. and Larson, Matthew R. and Sibert, Bryan S. and Kim, Joseph Y. and Parrell, Daniel and Sanchez, Juan C. and Pappas, Victoria and Kumar, Anil and Cai, Kai and Thompson, Keith and Wright, Elizabeth R.},
title = {Correlative montage parallel array cryo-tomography for in situ structural cell biology.},
journal = {Nature methods},
year = {2023},
volume = {20},
pages = {1537--1543},
doi = {10.1038/s41592-023-01999-5}
}
|
| Eisenstein F, Yanagisawa H, Kashihara H, Kikkawa M, Tsukita S and Danev R (2023), "Parallel cryo electron tomography on in situ lamellae.", Nature methods. Vol. 20, pp. 131-138. |
| Abstract: In situ cryo electron tomography of cryo focused ion beam milled samples has emerged in recent years as a powerful technique for structural studies of macromolecular complexes in their native cellular environment. However, the possibilities for recording tomographic tilt series in a high-throughput manner are limited, in part by the lamella-shaped samples. Here we utilize a geometrical sample model and optical image shift to record tens of tilt series in parallel, thereby saving time and gaining access to sample areas conventionally used for tracking specimen movement. The parallel cryo electron tomography (PACE-tomo) method achieves a throughput faster than 5 min per tilt series and allows for the collection of sample areas that were previously unreachable, thus maximizing the amount of data from each lamella. Performance testing with ribosomes in vitro and in situ on state-of-the-art and general-purpose microscopes demonstrated the high throughput and quality of PACE-tomo. |
BibTeX:
@article{Eisenstein2023,
author = {Eisenstein, Fabian and Yanagisawa, Haruaki and Kashihara, Hiroka and Kikkawa, Masahide and Tsukita, Sachiko and Danev, Radostin},
title = {Parallel cryo electron tomography on in situ lamellae.},
journal = {Nature methods},
year = {2023},
volume = {20},
pages = {131--138},
doi = {10.1038/s41592-022-01690-1}
}
|
| Gilbert MAG, Fatima N, Jenkins J, O’Sullivan TJ, Schertel A, Halfon Y, Morrema THJ, Geibel M, Radford SE, Hoozemans JJM and Frank RAW (2023), "In situcryo-electron tomography of β-amyloid and tau in post-mortem Alzheimer’s disease brain" Cold Spring Harbor Laboratory. |
BibTeX:
@article{Gilbert2023,
author = {Gilbert, Madeleine A. G. and Fatima, Nayab and Jenkins, Joshua and O’Sullivan, Thomas J. and Schertel, Andreas and Halfon, Yehuda and Morrema, Tjado H. J. and Geibel, Mirjam and Radford, Sheena E. and Hoozemans, Jeroen J. M. and Frank, René A. W.},
title = {In situcryo-electron tomography of β-amyloid and tau in post-mortem Alzheimer’s disease brain},
publisher = {Cold Spring Harbor Laboratory},
year = {2023},
doi = {10.1101/2023.07.17.549278}
}
|
| Zhao C, Lu D, Zhao Q, Ren C, Zhang H, Zhai J, Gou J, Zhu S, Zhang Y and Gong X (2023), "Computational methods for in situ structural studies with cryogenic electron tomography", Frontiers in Cellular and Infection Microbiology. Vol. 13 Frontiers Media SA. |
BibTeX:
@article{Zhao2023,
author = {Zhao, Cuicui and Lu, Da and Zhao, Qian and Ren, Chongjiao and Zhang, Huangtao and Zhai, Jiaqi and Gou, Jiaxin and Zhu, Shilin and Zhang, Yaqi and Gong, Xinqi},
title = {Computational methods for in situ structural studies with cryogenic electron tomography},
journal = {Frontiers in Cellular and Infection Microbiology},
publisher = {Frontiers Media SA},
year = {2023},
volume = {13},
doi = {10.3389/fcimb.2023.1135013}
}
|
| Gorbea Colón JJ, Palao L, Chen S-F, Kim HJ, Snyder L, Chang Y-W, Tsai K-L and Murakami K (2023), "Structural basis of a transcription pre-initiation complex on a divergent promoter", Molecular Cell. Vol. 83(4), pp. 574-588.e11. Elsevier BV. |
BibTeX:
@article{GorbeaColon2023,
author = {Gorbea Colón, Jose J. and Palao, Leon and Chen, Shin-Fu and Kim, Hee Jong and Snyder, Laura and Chang, Yi-Wei and Tsai, Kuang-Lei and Murakami, Kenji},
title = {Structural basis of a transcription pre-initiation complex on a divergent promoter},
journal = {Molecular Cell},
publisher = {Elsevier BV},
year = {2023},
volume = {83},
number = {4},
pages = {574--588.e11},
doi = {10.1016/j.molcel.2023.01.011}
}
|
| Kaplan M, Chang Y-W, Oikonomou CM, Nicolas WJ, Jewett AI, Kreida S, Dutka P, Rettberg LA, Maggi S and Jensen GJ (2023), "Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography.", Nature microbiology. Vol. 8, pp. 1267-1279. |
| Abstract: Bdellovibrio bacteriovorus is a microbial predator that offers promise as a living antibiotic for its ability to kill Gram-negative bacteria, including human pathogens. Even after six decades of study, fundamental details of its predation cycle remain mysterious. Here we used cryo-electron tomography to comprehensively image the lifecycle of B. bacteriovorus at nanometre-scale resolution. With high-resolution images of predation in a native (hydrated, unstained) state, we discover several surprising features of the process, including macromolecular complexes involved in prey attachment/invasion and a flexible portal structure lining a hole in the prey peptidoglycan that tightly seals the prey outer membrane around the predator during entry. Unexpectedly, we find that B. bacteriovorus does not shed its flagellum during invasion, but rather resorbs it into its periplasm for degradation. Finally, following growth and division in the bdelloplast, we observe a transient and extensive ribosomal lattice on the condensed B. bacteriovorus nucleoid. |
BibTeX:
@article{Kaplan2023,
author = {Kaplan, Mohammed and Chang, Yi-Wei and Oikonomou, Catherine M. and Nicolas, William J. and Jewett, Andrew I. and Kreida, Stefan and Dutka, Przemysław and Rettberg, Lee A. and Maggi, Stefano and Jensen, Grant J.},
title = {Bdellovibrio predation cycle characterized at nanometre-scale resolution with cryo-electron tomography.},
journal = {Nature microbiology},
year = {2023},
volume = {8},
pages = {1267--1279},
doi = {10.1038/s41564-023-01401-2}
}
|
| Hover S, Charlton FW, Hellert J, Swanson JJ, Mankouri J, Barr JN and Fontana J (2023), "Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K (+)during entry.", Nature communications. Vol. 14, pp. 5885. |
| Abstract: Following endocytosis, enveloped viruses employ the changing environment of maturing endosomes as cues to promote endosomal escape, a process often mediated by viral glycoproteins. We previously showed that both high [K (+)] and low pH promote entry of Bunyamwera virus (BUNV), the prototypical bunyavirus. Here, we use sub-tomogram averaging and AlphaFold, to generate a pseudo-atomic model of the whole BUNV glycoprotein envelope. We unambiguously locate the Gc fusion domain and its chaperone Gn within the floor domain of the spike. Furthermore, viral incubation at low pH and high [K (+)], reminiscent of endocytic conditions, results in a dramatic rearrangement of the BUNV envelope. Structural and biochemical assays indicate that pH 6.3/K (+)in the absence of a target membrane elicits a fusion-capable triggered intermediate state of BUNV GPs; but the same conditions induce fusion when target membranes are present. Taken together, we provide mechanistic understanding of the requirements for bunyavirus entry. |
BibTeX:
@article{Hover2023,
author = {Hover, Samantha and Charlton, Frank W. and Hellert, Jan and Swanson, Jessica J. and Mankouri, Jamel and Barr, John N. and Fontana, Juan},
title = {Organisation of the orthobunyavirus tripodal spike and the structural changes induced by low pH and K (+)during entry.},
journal = {Nature communications},
year = {2023},
volume = {14},
pages = {5885},
doi = {10.1038/s41467-023-41205-w}
}
|
| Pacheco-Fiallos B, Vorländer MK, Riabov-Bassat D, Fin L, O'Reilly FJ, Ayala FI, Schellhaas U, Rappsilber J and Plaschka C (2023), "mRNA recognition and packaging by the human transcription-export complex.", Nature. Vol. 616, pp. 828-835. |
| Abstract: Newly made mRNAs are processed and packaged into mature ribonucleoprotein complexes (mRNPs) and are recognized by the essential transcription-export complex (TREX) for nuclear export (1,2). However, the mechanisms of mRNP recognition and three-dimensional mRNP organization are poorly understood (3). Here we report cryo-electron microscopy and tomography structures of reconstituted and endogenous human mRNPs bound to the 2-MDa TREX complex. We show that mRNPs are recognized through multivalent interactions between the TREX subunit ALYREF and mRNP-bound exon junction complexes. Exon junction complexes can multimerize through ALYREF, which suggests a mechanism for mRNP organization. Endogenous mRNPs form compact globules that are coated by multiple TREX complexes. These results reveal how TREX may simultaneously recognize, compact and protect mRNAs to promote their packaging for nuclear export. The organization of mRNP globules provides a framework to understand how mRNP architecture facilitates mRNA biogenesis and export. |
BibTeX:
@article{PachecoFiallos2023,
author = {Pacheco-Fiallos, Belén and Vorländer, Matthias K. and Riabov-Bassat, Daria and Fin, Laura and O'Reilly, Francis J. and Ayala, Farja I. and Schellhaas, Ulla and Rappsilber, Juri and Plaschka, Clemens},
title = {mRNA recognition and packaging by the human transcription-export complex.},
journal = {Nature},
year = {2023},
volume = {616},
pages = {828--835},
doi = {10.1038/s41586-023-05904-0}
}
|
| Mühleip A, Flygaard RK, Baradaran R, Haapanen O, Gruhl T, Tobiasson V, Maréchal A, Sharma V and Amunts A (2023), "Structural basis of mitochondrial membrane bending by the I-II-III (2)-IV (2)supercomplex.", Nature. Vol. 615, pp. 934-938. |
| Abstract: Mitochondrial energy conversion requires an intricate architecture of the inner mitochondrial membrane (1). Here we show that a supercomplex containing all four respiratory chain components contributes to membrane curvature induction in ciliates. We report cryo-electron microscopy and cryo-tomography structures of the supercomplex that comprises 150 different proteins and 311 bound lipids, forming a stable 5.8-MDa assembly. Owing to subunit acquisition and extension, complex I associates with a complex IV dimer, generating a wedge-shaped gap that serves as a binding site for complex II. Together with a tilted complex III dimer association, it results in a curved membrane region. Using molecular dynamics simulations, we demonstrate that the divergent supercomplex actively contributes to the membrane curvature induction and tubulation of cristae. Our findings highlight how the evolution of protein subunits of respiratory complexes has led to the I-II-III (2)-IV (2)supercomplex that contributes to the shaping of the bioenergetic membrane, thereby enabling its functional specialization. |
BibTeX:
@article{Muehleip2023,
author = {Mühleip, Alexander and Flygaard, Rasmus Kock and Baradaran, Rozbeh and Haapanen, Outi and Gruhl, Thomas and Tobiasson, Victor and Maréchal, Amandine and Sharma, Vivek and Amunts, Alexey},
title = {Structural basis of mitochondrial membrane bending by the I-II-III (2)-IV (2)supercomplex.},
journal = {Nature},
year = {2023},
volume = {615},
pages = {934--938},
doi = {10.1038/s41586-023-05817-y}
}
|
| Yanagisawa H, Kita Y, Oda T and Kikkawa M (2023), "Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane.", Communications biology. Vol. 6, pp. 1018. |
| Abstract: The urothelium, a distinct epithelial tissue lining the urinary tract, serves as an essential component in preserving urinary tract integrity and thwarting infections. The asymmetric unit membrane (AUM), primarily composed of the uroplakin complex, constitutes a critical permeability barrier in fulfilling this role. However, the molecular architectures of both the AUM and the uroplakin complex have remained enigmatic due to the paucity of high-resolution structural data. In this study, we utilized cryo-electron microscopy to elucidate the three-dimensional structure of the uroplakin complex within the porcine AUM. While the global resolution achieved was 3.5 Å, we acknowledge that due to orientation bias, the resolution in the vertical direction was determined to be 6.3 Å. Our findings unveiled that the uroplakin complexes are situated within hexagonally arranged crystalline lipid membrane domains, rich in hexosylceramides. Moreover, our research rectifies a misconception in a previous model by confirming the existence of a domain initially believed to be absent, and pinpointing the accurate location of a crucial Escherichia coli binding site implicated in urinary tract infections. These discoveries offer valuable insights into the molecular underpinnings governing the permeability barrier function of the urothelium and the orchestrated lipid phase formation within the plasma membrane. |
BibTeX:
@article{Yanagisawa2023,
author = {Yanagisawa, Haruaki and Kita, Yoshihiro and Oda, Toshiyuki and Kikkawa, Masahide},
title = {Cryo-EM elucidates the uroplakin complex structure within liquid-crystalline lipids in the porcine urothelial membrane.},
journal = {Communications biology},
year = {2023},
volume = {6},
pages = {1018},
doi = {10.1038/s42003-023-05393-x}
}
|
| Kaplan M, Yao Q and Jensen GJ (2023), "Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography", International Journal of Molecular Sciences. Vol. 24(9), pp. 8292. MDPI AG. |
BibTeX:
@article{Kaplan2023a,
author = {Kaplan, Mohammed and Yao, Qing and Jensen, Grant J.},
title = {Structure and Assembly of the Proteus mirabilis Flagellar Motor by Cryo-Electron Tomography},
journal = {International Journal of Molecular Sciences},
publisher = {MDPI AG},
year = {2023},
volume = {24},
number = {9},
pages = {8292},
doi = {10.3390/ijms24098292}
}
|
| Digel L, Justesen ML, Bonné R, Fransaert N, Wouters K, Jensen PB, Plum-Jensen LE, Marshall IPG, Nicolas-Asselineau L, Drace T, Bøggild A, Hansen JL, Schramm A, Bøjesen ED, Nielsen LP, Manca JV and Boesen T (2023), "Comparative electric and ultrastructural studies of cable bacteria reveal new components of conduction machinery" Cold Spring Harbor Laboratory. |
BibTeX:
@article{Digel2023,
author = {Digel, Leonid and Justesen, Mads L. and Bonné, Robin and Fransaert, Nico and Wouters, Koen and Jensen, Pia B. and Plum-Jensen, Lea E. and Marshall, Ian P. G. and Nicolas-Asselineau, Louison and Drace, Taner and Bøggild, Andreas and Hansen, John L. and Schramm, Andreas and Bøjesen, Espen D. and Nielsen, Lars P. and Manca, Jean V. and Boesen, Thomas},
title = {Comparative electric and ultrastructural studies of cable bacteria reveal new components of conduction machinery},
publisher = {Cold Spring Harbor Laboratory},
year = {2023},
doi = {10.1101/2023.05.24.541955}
}
|
| Hwang JY, Chai P, Nawaz S, Choi J, Lopez-Giraldez F, Hussain S, Bilguvar K, Mane S, Lifton RP, Ahmad W, Zhang K and Chung J-J (2023), "LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility" Cold Spring Harbor Laboratory. |
BibTeX:
@article{Hwang2023a,
author = {Hwang, Jae Yeon and Chai, Pengxin and Nawaz, Shoaib and Choi, Jungmin and Lopez-Giraldez, Francesc and Hussain, Shabir and Bilguvar, Kaya and Mane, Shrikant and Lifton, Richard P. and Ahmad, Wasim and Zhang, Kai and Chung, Jean-Ju},
title = {LRRC23 truncation impairs radial spoke 3 head assembly and sperm motility underlying male infertility},
publisher = {Cold Spring Harbor Laboratory},
year = {2023},
doi = {10.1101/2023.02.25.530050}
}
|
| Draganova EB, Wang H, Wu M, Liao S, Vu A, Gonzalez-Del Pino GL, Zhou ZH, Roller RJ and Heldwein EE (2023), "The universal suppressor mutation in the HSV-1 nuclear egress complex restores membrane budding defects by stabilizing the oligomeric lattice" Cold Spring Harbor Laboratory. |
BibTeX:
@article{Draganova2023,
author = {Draganova, Elizabeth B. and Wang, Hui and Wu, Melanie and Liao, Shiqing and Vu, Amber and Gonzalez-Del Pino, Gonzalo L. and Zhou, Z. Hong and Roller, Richard J. and Heldwein, Ekaterina E.},
title = {The universal suppressor mutation in the HSV-1 nuclear egress complex restores membrane budding defects by stabilizing the oligomeric lattice},
publisher = {Cold Spring Harbor Laboratory},
year = {2023},
doi = {10.1101/2023.06.22.546118}
}
|
| Pražák V, Mironova Y, Vasishtan D, Hagen C, Laugks U, Jensen Y, Sanders S, Heumann JM, Bosse JB, Klupp B, Mettenleiter TC, Grange M and Grünewald K (2023), "NECing goes: flexibility of the herpesvirus nuclear egress complex" Cold Spring Harbor Laboratory. |
BibTeX:
@article{Prazak2023,
author = {Pražák, Vojtěch and Mironova, Yuliia and Vasishtan, Daven and Hagen, Christoph and Laugks, Ulrike and Jensen, Yannick and Sanders, Saskia and Heumann, John M. and Bosse, Jens B. and Klupp, Barbara and Mettenleiter, Thomas C. and Grange, Michael and Grünewald, Kay},
title = {NECing goes: flexibility of the herpesvirus nuclear egress complex},
publisher = {Cold Spring Harbor Laboratory},
year = {2023},
doi = {10.1101/2023.07.07.547920}
}
|
| Creekmore BC, Kixmoeller K, Black BE, Lee EB and Chang Y-W (2023), "Native ultrastructure of fresh human brain vitrified directly from autopsy revealed by cryo-electron tomography with cryo-plasma focused ion beam milling" Cold Spring Harbor Laboratory. |
BibTeX:
@article{Creekmore2023,
author = {Creekmore, Benjamin C. and Kixmoeller, Kathryn and Black, Ben E. and Lee, Edward B. and Chang, Yi-Wei},
title = {Native ultrastructure of fresh human brain vitrified directly from autopsy revealed by cryo-electron tomography with cryo-plasma focused ion beam milling},
publisher = {Cold Spring Harbor Laboratory},
year = {2023},
doi = {10.1101/2023.09.13.557623}
}
|
| Tan ZY, Cai S, Noble AJ, Chen JK, Shi J and Gan L (2023), "Heterogeneous non-canonical nucleosomes predominate in yeast cells in situ", eLife. Vol. 12 eLife Sciences Publications, Ltd. |
BibTeX:
@article{Tan2023,
author = {Tan, Zhi Yang and Cai, Shujun and Noble, Alex J and Chen, Jon K and Shi, Jian and Gan, Lu},
title = {Heterogeneous non-canonical nucleosomes predominate in yeast cells in situ},
journal = {eLife},
publisher = {eLife Sciences Publications, Ltd},
year = {2023},
volume = {12},
doi = {10.7554/elife.87672.3}
}
|
| Sibert BS, Kim JY, Yang JE, Hannon-Hatfield A, Ke Z, Garfinkel DJ and Wright ER (2023), "Workflow for High-resolution Sub-volume Averaging from Heterogenous Viral and Virus-like Assemblies", Microscopy and Microanalysis. Vol. 29(Supplement_1), pp. 943-944. Oxford University Press (OUP). |
BibTeX:
@article{Sibert2023,
author = {Sibert, Bryan S and Kim, Joseph Y and Yang, Jae E and Hannon-Hatfield, Adam and Ke, Zunlong and Garfinkel, David J and Wright, Elizabeth R},
title = {Workflow for High-resolution Sub-volume Averaging from Heterogenous Viral and Virus-like Assemblies},
journal = {Microscopy and Microanalysis},
publisher = {Oxford University Press (OUP)},
year = {2023},
volume = {29},
number = {Supplement_1},
pages = {943--944},
doi = {10.1093/micmic/ozad067.470}
}
|
| Farci D, Milenkovic S, Iesu L, Tanas M, Ceccarelli M and Piano D (2023), "Structural characterization and functional insights into the Type II Secretion System of the poly-extremophile Deinococcus radiodurans", Journal of Biological Chemistry., pp. 105537. Elsevier BV. |
BibTeX:
@article{Farci2023,
author = {Farci, Domenica and Milenkovic, Stefan and Iesu, Luca and Tanas, Marta and Ceccarelli, Matteo and Piano, Dario},
title = {Structural characterization and functional insights into the Type II Secretion System of the poly-extremophile Deinococcus radiodurans},
journal = {Journal of Biological Chemistry},
publisher = {Elsevier BV},
year = {2023},
pages = {105537},
doi = {10.1016/j.jbc.2023.105537}
}
|
| Chen L, Fukata Y and Murata K (2023), "In situ cryo-electron tomography: a new method to elucidate cytoplasmic zoning at the molecular level", The Journal of Biochemistry. Oxford University Press (OUP). |
BibTeX:
@article{Chen2023a,
author = {Chen, Lin and Fukata, Yuko and Murata, Kazuyoshi},
title = {In situ cryo-electron tomography: a new method to elucidate cytoplasmic zoning at the molecular level},
journal = {The Journal of Biochemistry},
publisher = {Oxford University Press (OUP)},
year = {2023},
doi = {10.1093/jb/mvad102}
}
|
| Ferreira JL, Pražák V, Vasishtan D, Siggel M, Hentzschel F, Binder AM, Pietsch E, Kosinski J, Frischknecht F, Gilberger TW and Grünewald K (2023), "Variable microtubule architecture in the malaria parasite.", Nature communications. Vol. 14, pp. 1216. |
| Abstract: Microtubules are a ubiquitous eukaryotic cytoskeletal element typically consisting of 13 protofilaments arranged in a hollow cylinder. This arrangement is considered the canonical form and is adopted by most organisms, with rare exceptions. Here, we use in situ electron cryo-tomography and subvolume averaging to analyse the changing microtubule cytoskeleton of Plasmodium falciparum, the causative agent of malaria, throughout its life cycle. Unexpectedly, different parasite forms have distinct microtubule structures coordinated by unique organising centres. In merozoites, the most widely studied form, we observe canonical microtubules. In migrating mosquito forms, the 13 protofilament structure is further reinforced by interrupted luminal helices. Surprisingly, gametocytes contain a wide distribution of microtubule structures ranging from 13 to 18 protofilaments, doublets and triplets. Such a diversity of microtubule structures has not been observed in any other organism to date and is likely evidence of a distinct role in each life cycle form. This data provides a unique view into an unusual microtubule cytoskeleton of a relevant human pathogen. |
BibTeX:
@article{Ferreira2023,
author = {Ferreira, Josie L. and Pražák, Vojtěch and Vasishtan, Daven and Siggel, Marc and Hentzschel, Franziska and Binder, Annika M. and Pietsch, Emma and Kosinski, Jan and Frischknecht, Friedrich and Gilberger, Tim W. and Grünewald, Kay},
title = {Variable microtubule architecture in the malaria parasite.},
journal = {Nature communications},
year = {2023},
volume = {14},
pages = {1216},
doi = {10.1038/s41467-023-36627-5}
}
|