Guide to Particle Estimation for Electron Tomography (PEET)

Boulder Laboratory for 3-D Electron Microscopy of Cells


Table of Contents


1. Introduction to PEET
2. The Setup Tab
2.1. Root Name for Output Field
2.2. Directory Field
2.3.Import an Existing Project Button
2.4. Copy Parameters Button
2.5. Volume Table
2.6. Using Relative Orientations
2.7. Action Buttons
2.8. Reference Section
2.9. Missing Wedge Compensation Section
2.10. Masking Section
2.11. Initial Motive List Section
2.12. Y Axis Type Section
3. The Run Tab
3.1. Parallel Processing
3.2. Iteration Table
3.3. Spherical Sampling for Theta and Psi Section
3.4. Remove duplicates
3.5. Particle Volume Fields
3.6. Use Equal Numbers of Particles Checkboxes
3.7. Number of Particles in Averages Section
3.8. Align averages to have their Y axes vertical
3.9. Particles per CPU
3.10. Action Buttons
3.11. Advanced Options


1. Introduction to PEET

PEET is a set of programs, separate from IMOD, to align and average particles extracted from 3D volumes.  It finds the optimal alignment of each particle with a reference volume through several iterations.  Subsequent iterations refine the optimal alignment found in the previous iteration by aligning to a new reference generated from some of the particles aligned on the previous iteration.  It has a graphical user interface that helps users to set up options (working directory, tomogram file name, searching parameters, etc.), and to control the execution of the process.  This guide will explain each option in the interface.

The interface can be accessed by starting eTomo and pressing the New PEET button, or by selecting File-->New-->PEET in the menu.  The PEET panel has 2 tabs, the Setup tab and the Run tab.  The Setup tab has options for defining the data to be worked on and setting up initial conditions and other features of the search.  The Run tab has a table for entering parameters to control each iteration of the search and options for the final averaging operation.

2. The Setup Tab

2.1. Root Name for Output Field

Specify the base name that PEET will use to name the files it creates.  To make this manual concrete, we will assume a base name of "myRun".

2.2. Directory Field

Push the open file icon to pick or create a working directory.  PEET will store temporary files and final average volumes in this directory.  A new directory is required for each separate PEET run, because a large number of files can be created.  It is not necessary to copy or move tomograms and models into the data directory.

Assuming a base name of "myRun", some of the important text files in this directory are listed in the table below,  (Here, #k and #m stand for integers, and "*" is a wildcard which can match any series of characters.)

myRun.epe

The project file, containing eTomo status and other options appearing on the screen but not stored in the PEET parameter file

myRun.prm

The parameter file, containing PEET parameter assignments in Matlab syntax.  See the PEET man page for additional details.

myRun*.com

Command scripts that will distributed to user-selected computers for execution.

myRun_MOTL_Tom#k_Iter#m.csv

Optimal alignments found for tomogram #k particles during alignment iteration #m - 1.  PEET uses the contents as inputs subject to further refinement in iteration #m.  These files are in "comma separated value" (.csv) format, so they can be examined or modified in text editors or in spreadsheet programs like Excel or OpenOffice Calc.  "MOTL" stands for motive list, a set of rotations, translations, and other information about each particle, including the correlation coefficient from the alignment with the reference.

myRun*.log

Logs created by the shell commands.  Each .com file creates a corresponding log file.


Files with .mrc extension in this folder are volumes stored in MRC format.  References and final averages are examples of important volumes created by PEET and stored in this format.  For example, myRun_Ref#m.mrc is the reference volume created for alignment during iteration #m.  Similarly, the average volume resulting from combining #n particles at iteration #m is stored in myRun_AvgVol_#mP#n.mrc.

Once you have defined the root name and the working directory, you can begin to enter data into the volume table.  In addition, before you have any data, you have a chance to bring parameter entries into this new project in two different ways, described next.

2.3. Import an Existing Project Button

Use contents of an existing .prm or.epe file to fill in parameters, including the volume table.  The main uses for importing a .prm file are 1) if you have processed data with PEET outside of the eTomo graphical interface, and want to create an eTomo project; 2) if you have a master parameter file that you want to copy settings from; or 3) if you want to make a duplicate of an existing project and run it again with different parameters.  Importing an .epe file also imports its corresponding .prm file.

2.4. Copy Parameters Button

Copy the Iteration Table and other parameters not relating to specific volumes from an existing project or parameter file.  The Volume table and references to specific model, object, contour, or particle numbers will not be copied.  This control is useful, for example, if you would like to transfer search parameters used previously to a new project using different volumes.

2.5. Volume Table

The Volume Table can have multiple rows, each referring to a volume and corresponding model to be included in the alignment.  A row can be selected by clicking => near the left end of the row.  Each row can contain 8 entries, described in the following table. 

Volume

The name of a file containing a tomogram in MRC format and oriented so that X / Y planes correspond to the plane of the specimen.  (Note that post-processing will typically be necessary after reconstruction to meet this requirement.)  The same volume can be entered on more than one row.

Model

The name of a file containing an IMOD model for the current volume.  All points in the first object of the model, and only those points, will be used, with each point specifying the center of a particle to be aligned and averaged.  (Points in objects other than the first can be useful for specifying orientation in some cases.  See Y Axis Type below.)  Typically, the object will contain either scattered points for isolated particles, or open contours if the particles lie along filaments, and will be assigned the corresponding type.

Initial MOTL

The name of a file containing an initial motive list, specifying rotations and / or translations required to approximately align each particle with the reference.  This can permit use of a more restricted search for the optimal alignment.  The file format is identical to that of the .csv MOTL files used by PEET to transfer alignment information between iterations.  This parameter is active only if the Use files option is selected in the "Initial Motive List" section, described below.

Tilt Range

The tilt range used to collect the tomogram, which is needed to compensate averages and correlation coefficients for the effects of the tomographic "missing wedge".  This option is disabled by default.  To enable it, check Use tilt range in averaging in the "Missing Wedge Compensation" checkbox group.  If you have a file with tilt angles for this tomogram, you can transfer the appropriate values from the file to the table by pressing the Read Tilt File button beneath the table.  The default type of file is a "tilt.log", whose angles will always be correct. You can also change the filter in the file chooser to select a ".tlt" file, whose angles will be correct as long as the no additional angle offset was entered when running the Tilt program.  Tilt range must be specified in order to use missing wedge compensation.

Rel. Orient.

Relative orientation consists of 3 angles representing a rotation that will be applied to each particle in the tomogram to bring it to a standard, starting orientation, ideally that of the reference.  This rotation is followed by any rotations specified for individual particles in the initial motive list.  Relative orientation angles are 3dmod slicer X, Y, and Z angles in degrees, and are applied in the order Z, Y, X.  The use of this feature is described below in more detail.


2.6. Using Relative Orientations

The most common use of relative orientations is when combining data from multiple averages with different orientations from prior PEET runs on separate volumes.  In this situation, you might pick the final reference from one of the tomograms as the reference for further alignment.  Use the final MOTL files from the previous runs as the initial MOTL files for each volume.  Open the final reference from each of the other tomograms in 3dmod and rotate it in Slicer until it approximately matches the orientation of the chosen reference.  One way to do this is to open all of the final references together in 3dmod and use Edit-Angles to open the Slicer Angles dialog.  After you turn on "Auto" in the toolbar that appears in the Slicer, angles are continuously saved in the Angles table as you change them in the Slicer, and when you switch between different volumes (times) the saved angles will be restored for the volume that you switch to.  In this way, you can adjust angles and toggle between volumes to assess the alignment.

When aligning the volumes from the different tomograms in this way, you may want to rotate the chosen reference as well as all the others into a more convenient orientation.  If you do this, the alignment search with all of the tomograms will work correctly if you specify a particle from one tomogram as the reference.  However, if you specify a file as the reference, the search will not work unless you also rotate the the chosen reference into the new orientation.  You can do this with the IMOD program rotatevol, e.g, with "rotatevol -ang Z,Y,X input_file output_file", where X, Y, and Z are the slicer angles entered as the relative orientation for the tomogram from which the reference was taken.

Another possible use of this feature would be to indicate relative orientations between the structures in multiple tomograms where no initial averaging has been done.  In this case, the reference could be a particle from one of the tomograms or a file with a reference volume.  Slicer angles should be found that bring the structures in a given tomogram into approximately the same orientation as this reference.  Relative orientation may also optionally be specified for the volume containing the reference.  In this case, the reference volume relative orientation can be thought of as reorienting the reference to a preferred orientation, while the relative orientations for other volumes are those required to match the reoriented reference.  As indicated in the table above, any rotations in an initial MOTL file will be applied after the rotation from the relative orientation.  Note, however, that if you have structures along fibers in multiple tomograms, it is preferable to use the Initial Motive List options described below to initialize the Z axis or the X and Z axes in the motive list; in these cases relative orientations are both unneeded and counterproductive.

2.7. Action Buttons

Press Add Volume and Model to add a row to the table.  File choosers will open to allow you to pick the volume and model files.  To add a row with the same volume, model, and other settings as in an existing row, highlight the existing row with => and press Copy Row.

After you highlight an existing row in the table with =>, other buttons become available.  Press Copy Row to duplicate an existing row, omitting relative orientations.  Press Change Model to change just the model file on the selected row.  Press Set Initial Motive List File to open a file chooser and select an initial MOTL file.  After enabling missing wedge compensation, pressing Read Tilt File will read the minimum and maximum tilt angles from a .tlt file.  (This option is available only if Initial Motive List has been set to Use files... see below).  Press Move Up or Move Down to change the location of the highlighted row in the table.  Press Delete Row to delete the current row from the table.  Press Open in 3dmod to open the volume and model files in 3dmod.  You can also right-click this button to open the volume binned by 2 or to open 3dmod with the startup window shown in order to pick other options.

2.8. Reference Section

Check the Volume radio button to use a particle from one of the tomograms in the Volume Table as the reference.  Input the tomogram number in the spin box and the particle number in the field with the heading Particle #.  Particles are numbered sequentially from 1 as if all the contours were combined into one; e.g., if the first contour has 100 particles, then the 20th particle of the second contour is number 120.

Check the Reference file radio button to use a separate volume, specified by file name, as the reference and input the file name of the stack in the appropriate field or use the file chooser to select it.

2.9. Missing Wedge Compensation Section

Check Use tilt range in averaging to use PEET's missing wedge compensation for averaging.  When selected, object space averaging of the aligned particles will be replaced by a weighted averaging in Fourier space, with each particle contributing only to regions in which its projections are informative, i.e. those lying outside its tomographic missing wedge.

If the first option is selected, you can also check Use tilt range in alignment to use PEET's missing wedge compensation of correlation coefficients during alignment.  This compensation involves boosting the correlation coefficients when there is less overlap between informative regions of the particle and the reference.  Specifically, the correlation coefficient is multiplied by the ratio between the maximum, over all particles, of the fraction of Fourier space having data from informative regions of both the particle and the reference and the fraction for the current particle at a specific orientation.  This kind of compensation is relevant only when the reference is a particle with a missing wedge rather than an average.

If Use tilt range in alignment is checked, a additional type of missing wedge compensation is available during averaging at the end of the first iteration.  After computing optimal alignments, particles are divided into groups based on the extent of overlap between each particle's informative area and that of the reference in Fourier space.  Correlation scores are then scaled so that each group has approximately the same median score.  The adjusted scores are then used to select particles to form the reference for the second iteration, as well as averages if the user chooses to create them at the end of the first iteration.  The result is that nearly equal numbers of particles from each of the orientation groups are included in the reference, thus minimizing a missing wedge bias in that reference that would be reinforced on the second iteration.  To enable this option, select # of weight groups for equalizing CCCs and choose the desired number of groups.  The number of groups to use depends on the number of particles.  It is probably best to have at least 20 particles per group, but there is little to be gained from having more than 10 groups even if there are many particles.  This option is most effective if particles assume a variety of original orientations.  If particles all have about the same orientation, (e.g. particles from a non-twisting microtubule sitting in the X-Y plane), this type of compensation may not be necessary or helpful.

The Edge shift entry specifies the number of pixels in from the edge of the missing wedge to include when averaging with missing wedge compensation.  Some useful information seems to spread into the missing wedge during backprojection.  A value of 1-2 pixels is appropriate.

2.10. Masking Section

Users can mask out an area of the reference that they want to exclude from being used for cross-correlation.  The default is no masking.  However, the user can choose a volume, a sphere, or a cylinder as the mask by checking the corresponding radio button.  For example, when averaging an icosahedral virus, you might use a spherical mask to align only on the capsid and mask out the interior of the virus, or vice versa.  A cylindrical mask could be used when aligning subunits of a microtubule to exclude variable material outside the microtubule, such as a neighboring microtubule.

To use a volume containing an arbitrary mask select the Volume option, and input the name of the file containing the mask volume.  A voxel of the reference volume will be masked out if the value of its corresponding voxel in the mask volume is zero.  The dimensions of the mask volume need not match those of the reference volume.  In this case, the mask volume is centered on the reference, and mask values outside the reference volume are ignored.  Mask values outside the mask volume, but within the reference, are determined by the value of the majority of the 8 corner voxels of the mask volume.  (I.e., if 5 or more corners are zero, the mask will be zero outside the volume;  conversely, if 4 or fewer corners are zero, the mask will be one outside the mask volume.)  A convenient way to generate a mask volume is to use the IMOD program imodmop, which can be used to retain pixels inside of spheres and cylinders as well as inside of closed contours.  For this purpose, it does not matter that this program leaves original density values rather than 1's in the region that is retained.

If the Sphere or Cylinder mask options are chosen, the user can specify both inner and outer radii.  Reference voxels inside the inner radius or outside the outer radius will be masked out.

A spherical mask will be centered on the center of the reference volume.

A cylindrical mask will be oriented with its long axis passing through the vector from the reference particle to the neighboring particle along the contour.  Alternatively, you can specify the orientation of a cylindrical mask by setting the model number and particle number in the Cylinder Orientation box. 

2.11. Initial Motive List Section

These controls provide several options for setting up an initial motive list with starting orientations for all particles.  Such initial orientations can allow the angular search to be done over a limited range, even when the particles have significantly different orientations.  The first, default option leaves all rotation angles zero, providing no orientation information, and, potentially, requiring a broad search. The next two options involve deriving an initial orientation from the line between one particle and its neighbor in the IMOD model.  When using either of these options, the reference should also be be specified by a volume and particle numbers (rather than file name), so that the orientation of the reference can be estimated in the same manner.  If you are modeling particles along more than one filament, each filament should be modeled in its own contour, so that PEET will not base any orientations on the difference in positions between the end of one filament and the start of the next. Finally, you can also choose to read initial motive list(s) from specified .csv files. In this case, translational shifts as well as orientation can be specified.

2.11.1. Set all rotational values to zero

All particles will have their initial orientation left unaltered. This is appropriate if the particles are scattered, independent particles, no prior alignment information is available, and the orientations are either not readily apparent from the images or you choose not to bother extracting this information, preferring instead to use a more extensive alignment search.

2.11.2. Initialize Z Axis

Use this option if each particle can be aligned approximately with the reference by rotating it only around the Z axis.  PEET will compute the rotations needed based on trajectories between successive points in the IMOD model.  We find the option is most useful when all particles have roughly the same Z coordinate (e.g. when particles are from a bending, non-twisting microtubule lying in the X-Y plane).

2.11.3. Initialize X and Z Axis

If the trajectory between particles does not lie in the X-Y plane, then this option can be used instead to compute a more general rotation that aligns the trajectory at a particle with that at the reference.

2.11.4. Use files

Use this option if you have a text file in .csv format specifying the approximate rotation (and, optionally, translation) required to orient each particle.  Such motive list (MOTL) files can be obtained from a previous run of PEET or from an auxiliary program that computes initial orientations from a model for a particular situation.  The PEET program modTwist2EM is an example of such a program which generates motive lists for particles from a bending, twisting microtubule assuming a general 3D orientation.

2.12. Y Axis Type Section

These options define the first rotation axis that PEET uses for angular search, the one referred to as Phi in the iteration table.  The default is the original Y axis of the tomogram.  In this case, Phi corresponds to the tomogram Y axis, Theta to the tomogram Z axis, and Psi to the tomogram X axis.  However, the user can define their own first rotation axis, and PEET will deduce the second and third rotation axis assuming a right-handed Cartesian coordinate system.  This option is useful when running searches with restricted ranges if you want to specify a different range or increment for the different axes.  It can be used both when the reference is a file as well as when it is a particle.

If Particle model points is selected, the first rotation axis will vary from particle to particle and will be the vector connecting 2 consecutive model points in the IMOD model contour.  With this option, Phi represents twisting around the contour axis; Theta represents bending or turning in the X-Y plane, and Psi represents dipping in Z.  This option is most useful when particles sit along contours, for example, when particles are along a microtubule.  When using this option, be sure to model separate filaments in separate contours.

If End points of contour is selected, an arbitrary vector in each volume can be used as the first rotation axis by taking the vector connecting the two end points of a contour in the model for that volume.  The contour is specified by two integers: the IMOD object number and the contour number.

3. The Run Tab

3.1. Parallel Processing

PEET can distribute the computational load of aligning and averaging particles over multiple processors on one or more systems, as specified by the user.  This section allows you to select which systems and how many processors on each system are to be used.  See the eTomo User's Guide for details on the controls in this section and on how to configure parallel processing on your system(s). For information on how to use a cluster queue, see the man pages for processchunks and the cpu.adoc file.

3.2. Iteration Table

Each row of this table specifies options for a single iteration and has five types of entries.

The Angular Search Range entries set search ranges and increments (step sizes) in degrees for the 3 rotations (Phi, Theta, and Psi, referring to rotations around Y, Z, and X axes).  For example, if Max and Incr fields of Phi are set to 6 and 2 respectively, Phi will have a range of -6 to +6 and will assume 7 values (-6, -4, -2, 0, 2, 4, and 6).  A general rule is that earlier iterations should have larger ranges and step sizes.  Additionally, multiple iterations with larger step sizes are typically faster than a single search with a large range but small size.  A reasonable compromise is to use a Max of 3 times Incr, as in the example above.  Both Max and Incr can then be reduced by a factor of 2 at each subsequent iteration, until the desired precision is achieved.  Searches with even fewer steps can be done by using a Max of twice the increment, provided that Max and Incr are reduced by a factor of only 1.5-1.6 between iterations.  In many cases, it may be necessary to use more evaluations during the first iteration to obtain a reasonable initial alignment.  To skip searching around a particular axis entirely, set the Max to 0 and Incr to 1.

Search Radius can be either a single number or three numbers, and specifies the amount of translation in pixels allowed in the X, Y, and Z directions during alignment searches.  For example, setting it to 2 will limit the translation to between -2 and +2 in each of the 3 directions.  These directions are in the coordinate system of the tomogram, i.e., of the unrotated particle, so using three different limits would be useful only if particles all had similar orientations with respect to at least one axis.  This entry does not affect execution time, but small values can make the alignment more reliable by preventing spurious correlation peaks at higher translations from being chosen.  Also, before correlation, regions as wide as the search radius at each edge of the particle volume are set to zero to avoid wraps around effects.  Thus, the larger the search radius, the fewer voxels are actually included in the correlation and the lower the signal-to-noise ratio of the correlation.  A search radius of 0 can be specified to disable searching, allowing the quality of the initial alignment to be determined.

High Freq. Filter specifies parameters for filtering out high frequencies.  As in IMOD, frequencies are in reciprocal pixels, with 0.5 being the Nyquist frequency, and 0.86 corresponding to the highest frequency in the corners of 3D FFTs.  The Cutoff field sets the cutoff frequency, below which there is no attenuation, and above which there is progressively more attenuation. Above the cutoff, the response falls like the right half of a Gaussian with standard deviation given by the Sigma field.  Both the reference and the particle are filtered separately, so the correlation is essentially filtered twice.  One way to see what a reference or particle looks like with a particular filter is apply the same filter to a file with the IMOD program mtffilter, e.g., with the command "mtffilter -3d -low cutoff,sigma input_file output_file".  You can do this with the reference particle extracted by PEET, or you can extract a particle from the tomogram in 3dmod using the rubber band in the Zap window and the Extract entry in the File menu.

Reference Threshold controls how many particles are used to form the reference for the next iteration.  If a value larger than one is specified, it represents the number of particles to use, with higher correlation coefficients chosen preferentially.  A rule of thumb is to use two thirds of the total number of particles.  In some cases, however, you might want to include all particles on a first round to minimize any missing wedge bias in the new reference.  If a value less than one is specified, it represents a correlation coefficient threshold, above which particles will be included in computation of the new reference.  We have not found specifying the threshold in this manner particularly useful.

Duplicate Tolerance provides control over PEET's duplicate rejection logic.  When the search range is comparable to the spacing between particles, and especially when particles lie along filaments, an alignment search can sometimes result in mulitple particles pointing to essentially the same position and orientation.  If not corrected, these errors can bias subsequent references and averages, and can lead to overestimation of resolution using Fourier Shell Correlation.  If Remove Duplicates is checked (below the Spherical Sampling section), PEET will attempt to identify cases of duplication, and will remove offending particles from further averaging or consideration during the current iteration.  The Shift and Angular tolerances specify the maximum separation in integer pixels and degrees, respectively, at at which particles can be considered duplicates.  The shift tolerance is applied separately to each of the tomogram X, Y, and Z coordinates. Duplication is determined independently near the end of each iteration, so a particle ingored as a duplicate in one iteration is not necessarily excluded from later iterations.  A value of 0 for either or both of the angular and shift tolerances can be used to disable duplicate removal for the associated iteration.

3.3.Spherical Sampling for Theta and Psi Section

This option is designed to make full angular searching on the first iteration more efficient by sampling orientation space at relatively uniform intervals.  It is referred to as spherical sampling because the second and third search angles (Theta and Psi) are chosen to given approximately uniform spacing when represented on the surface of a sphere.  (Phi corresponds to rotation around the particle's twist or Y axis).  Simply varying both Theta and Psi with regular increments results in oversampling near the poles on the X axis.  With spherical sampling enabled, PEET avoids this oversampling.  If Full sphere is checked, PEET will sample the whole sphere.  If Half sphere is checked, PEET will only sample one hemisphere.  The Sample interval field specifies the Theta search interval in degrees, as well as the Psi search interval at the equator.  For a unit sample sphere, sample points are placed on latitude lines separated by the sample interval, with approximately 360 / Sample interval points along along each latitude line at the equator, decreasing with latitude to no more than a single point at each pole.  For either a full or half sphere angular search, enter a Max of 180, with an appropriate Incr for Phi in the Iteration Table, as well as the Sample interval for spherical samping.  When in doubt, it is typically reasonable to set Sample interval to the same value as the Phi search increment.

3.4. Remove duplicates

As described above, selecting this option enables PEET's duplicate removal logic with further control provided by the Duplicate Tolerances in the Iteration Table.

3.5. Particle Volume Fields

These fields set the X, Y, and Z dimensions of the particles.  For example, if X, Y, and Z are all set to 64, particle volumes will have dimensions of 64x64x64.  The center of a particle volume is the X, Y, and Z position of the particle in an IMOD model. 

Because the FFT routines from IMOD are being used, each dimension must be even and have prime factors no greater than 19; eTomo will check the values that you enter to make sure they meet this restriction. Specifically, of the even numbers up to 100, the sizes 46, 58, 62, 74, 82, 86, 92, and 94 are not allowed.

3.6. Use Equal Numbers of Particles Checkboxes

The checkboxes Use equal numbers of particles from all tomograms for new reference and Use equal numbers of particles from all tomograms for averages provide another tool for reducing bias when combining data from multiple tomograms and using a particle or average from one as a reference.  If checked, PEET will use equal numbers of particles from each tomogram to form the references or averages when possible.  (Unequal numbers may still be used if required to acheive the requested number of particles).  Otherwise, it will choose particles with the highest correlation scores regardless of which tomogram(s) they are in.

3.7. Number of Particles in Averages Section

Specify how many particles to use to form averages.  For example, if Start is set to 50, Incr is set to 50, End is set to 150, and Additional numbers is set to 165; there will be 4 final averages, based on 50, 100, 150 and 165 particles.  If End is not bigger than Start by a multiple of the increment (e.g., 160), the last number in the sequence will be the one smaller than End (e.g., 150).

The biggest number entered in these fields should be less than the total number of particles in all tomograms.  If used, Additional numbers should be in increasing order and all larger than End.

3.9. Align averages to have their Y axes vertical

If selected, average volumes will be reoriented to have their Y axis approximately vertical.  This is particularly useful for particles along a straight or slightly curved filament.  Note that the reorientation is applied only to the particles as they are averaged and is not incorporated into the motive list, so you cannot use such an average as a reference for another iteration of alignment starting with the motive list from the last iteration.  However, the reference volume produced on the last iteration is in the original orientation and can validly be used as a reference.

3.9. Particles per CPU

Specify how many particles each CPU should process in one "chunk".  The fewer particles per chunk, the more command and log files will be created.  However, having more particles per chunk can make the parallel processing less flexible should you need to kill or pause and resume the processing.  The default of 5 is a good choice for most purposes.

3.10. Action Buttons

After reviewing and selecting Systems and / or processors in the Processing Table, press Run to start the processing.  At this point, eTomo will write the parameter file and run the PEET program prmParser, producing a series of command (.com) files which will be executed by processchunks to carry out the computations requested.  The first "-start.com" command file initializes the motive lists and prepares the initial reference.  This can take some time when the reference is a particle, as the program determines how big to make the reference.  Numbered command files for aligning are run next, with a "-sync.com" file run at the end of each iteration to combine the results from the different chunks and compute a reference for the next iteration. After the final iteration, a "-finish.com" command file creates the resulting average volumes.

When the processing is completed, press Open Averaged Volumes in 3dmod to see all of the computed averages together in 3dmod.  The Isosurface window will be opened automatically.  Note that the isosurface threshold can be adjusted independently for each "time" (average).  You can also press Open Reference Files in 3dmod to see the references generated on each round.  The reference will be larger than the averages.  You can also select Remake Averaged Volumes to recompute averages using the existing alignment, e.g. after changing the number of particles to average or other settings.

3.11. Advanced Options

A few additional PEET options are available only in "advanced" mode.  To see if you are in advanced mode, look at the upper right of the Run tab.  If the small, rectangular button at this position is labeled "A", you are in basic mode, and pressing this button will switch to advanced mode, making the additional options visible. If the button is labeled "B", you are currently in advanced mode, and pressing the button will switch back to basic mode.

3.11.1 Cross correlation measure

PEET can compute cross-correlation in either of two ways.  Normalized cross-correlation is recommended, and can be chosen by selecting the radio button labeled True local correlation coefficientLocal energy normalized correlation is a less accurate approximation which may be adequate in some cases. In early PEET releases, computation of the local approximation was considerably faster than normalized cross correlation. This is no longer the case, so we recommend always selecting True local correlation

3.11.2 Mean fill

In some cases, particles of interest may lie partially outside their parent volume.  If Mean fill is checked, voxels in the missing region will be replaced with the mean of the data which are present.  We cannot think of any reason to uncheck this box.

3.11.3 Aligned base name

If a string is entered in the Aligned base name field, PEET will save volumes containing the individual aligned particles used to create averages.  For example, if Aligned base name is set to "aligned", a file aligned_tom1_P0002.mrc will contain the the second aligned particle from the first tomogram.

3.11.4 Low frequency filter

Just as PEET can be told to filter out high frequencies from both the reference and particles being aligned, it can also be told to filter out low frequencies.  To do so, enter a pair of numbers both greater than zero.  The first represents the frequency in inverse pixels, below which the signal will be attenuated.  The second represents the standard deviation of the Gaussian whose left half defines the shape of the resulting high-pass filter.  We have not found low frequency filtering to be useful, and do not recommend its use.

3.11.5 Debug level

Select a number between 0 and 3 to control the contents of the log files.  Higher numbers store progressively more information to the logs.