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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:
@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:
@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:
@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:
@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:
@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:
@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:
@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}
}
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}
}
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}
}
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:
@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, 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}
}
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:
@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:
@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:
@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}
}
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}
}
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:
@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}
}
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}
}
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}
}
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}
}
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}
}
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:
@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:
@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:
@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, 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}
}
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}
}
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:
@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:
@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}
}
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 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:
@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:
@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:
@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:
@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}
}
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}
}
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, 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, 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}
}
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}
}
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}
}
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}
}
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}
}
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}
}
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}
}
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:
@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}
}
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:
@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}
}
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}
}
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}
}
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 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:
@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:
@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}
}
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}
}