Guide to Particle Estimation for Electron Tomography (PEET)

University of Colorado, Boulder

Last Revised: 3 January, 2023

Table of Contents

1. Introduction to PEET
2. The Setup Tab
2.1. Root Name for Output
2.2. Directory
2.3. Volume Table
2.4. Action Buttons
2.5. Reference Section
2.6. Volume Size
2.7. Missing Wedge Compensation Section
2.8. Masking Section
2.9. Particle Y Axis Section
2.10. Initial Motive List Section
3. The Run Tab
3.1. Parallel Processing
3.2. Iteration Table
3.3. Spherical Sampling for Theta and Psi Section
3.4. Number of Particles to Average
3.5. Use Equal Numbers of Particles From All Tomograms
3.6. Action Buttons
4. The More Options Tab
4.1. Particle Selection
4.2. Alignment
4.3. Processing
5. Resampling Existing Volumes / Models

1. Introduction to PEET

PEET is a set of programs, separate from but used in conjunction with IMOD, to align and average particles / subvolumes extracted from 3D volumes.  It iteratively refines particle alignments against a reference volume over several iterations.  A new reference is typically generated from a subset of the aligned particles at the end of each iteration for use in the following iteration. IMOD's etomo graphical user interface permits users to set up options (search parameters, etc.), and to control the execution of the process.  This guide explains the use of that interface.  For details on individual PEET programs, please consult the program descriptions and manual pages accessible from the etomo's Help Menu, by running "PEETHelp", or online at  Manual pages are also available at a shell prompt using the "man" command.  (Program descriptions and manual pages will also be available locally on systems with Imod and PEET installed).

Each PEET project must reside in its own directory because of the potentially large number of intermediate and output files created. Input files (IMOD models and volumes, initial motive lists, etc.) can also reside in this directory, but are not required to.

To access the interface and create a new PEET project, run etomo and either press the Subvolume Averaging (PEET) button, or select File-->New-->Subvolume Averaging (PEET) from the menu.This will bring up a dialog allowing you to specify the directory where the new project is to be located, as well as a base or root name to be used in generating output and intermediate file names. This dialog will also allow you to copy all the settings from an existing project as a starting point for the new one.

Alternatively, to open an existing PEET project simply cd to the desired directory and run etomo with the name of the desired project file, e.g. "etomo myProject.epe", or run etomo with no arguments and select File-->Open from the menu. You can then browse to and select the desired project file. (PEET project files have a .epe suffix, while parameter settings are stored in a .prm parameter file, as described below).

The etomo PEET gui has 3 tabs, labeled Setup, Run, and More Options.  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. The More Options tab has additional options, including both common and advanced options. (A handful of advanced options can only accessed by manually editing the parameter file).

2. The Setup Tab

2.1. Root Name for Output

(Read only). Displays the base name that PEET will use to create output and intermediate files.  In the following examples, we will assume a base name of "myRun".

2.2. Directory

(Read only). Displays the directory containing the PEET project.

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.)


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


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


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


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.


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 created by PEET are important examples.  For example, myRun_Ref#m.mrc is the reference volume created for alignment during iteration #m.  Similarly, an average resulting from combining #n particles at iteration #m is stored in myRun_AvgVol_#mP#n.mrc.

2.3. 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 side of the row. Each row can contain 5 entries, described in the following table.


The name of a file containing a tomogram in MRC format, typically oriented so that X / Y planes correspond to slices of the specimen.  (Post-processing may be necessary after reconstruction achieve this orientation.)  The same volume can be entered on more than one row.


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.  (While not used during alignment, objects other than the first are used by some PEET modeling programs, such as meshInit, modTwist2EM, spikeInit, and stalkInit). For alignment, object 1 will typically contain either scattered points for isolated particles or open contours for particles along filaments.

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 User supplied csv files option is selected in the "Initial Motive List" section, described below.

Tilt Range

Two values defining the tilt range used to collect the tomogram. Tilt range is used to correct averages and alignments for the effects of the tomographic "missing wedge".  This option is disabled by default.  To enable it, check Enabled under "Missing Wedge Compensation".  If you have a tilt file containing the tilt angles for the currently selected tomogram, you can read the appropriate values from the file by pressing the Read Tilt File button beneath the table.  The default type of file is a "tilt.log", whose angles should 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 IMOD's Tilt program.  Tilt range must be specified in order to use missing wedge compensation.

If 2 or more tilt axes are in use (see 2.7. Missing Wedge Compensation Section), Tilt Range will be replaced by a single column labeled "Missing Wedge Mask".  This field should contain the name of a file containing the missing data mask(s) for this row of the volume table. 

2.4. Action Buttons

Up, Down, Delete, and Dup buttons to the right of the volume table allow modifying the volume table by moving, deleting, or duplicating a selected existing row.  To select a row press => near the left of the row. The Insert button adds a new, blank row a the bottom of the volume table. Volume, Model, and Initial MOTL fields for any row in the volue table can be entered or modified either by selecting the file browser (folder) icon to the right of the field, or by typing directly in the text box.  Tilt ranges can be entered or modified by typing in the appropriate text box, or by pressing Read tilt file if you have the an appropriate file.  Selecting a row of the volume table and pressing Open in 3dmod will open and display the corresponding volume, assuming it is the name of a single volume.  If File names are templates is selected, any or all of Volume, Model, Initial MOTL, and Missing Wedge Mask can be templates mapping to many files, rather than the name of a single file. For example v1-60.mrc would be expanded to 60 volumes: v1.mrc, v2.mrc, ... v60.mrc. 

2.5. Reference Section

The Reference section allows specification of the initial reference to be used for the first alignment iteration. If the Particle radio button is pressed, a single, specified particle from one of the input volumes will be used as the initial reference.  Tomograms and particles are both numbered sequentially from 1, and particles are numbered 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 will be particle number 120.  Note that PEET will align and average only particles and contours contained in the object 1.  Additional objects will be ignored during alignment and averaging, although they are used by some specific PEET programs (e.g. meshInit, modTwist2EM, spikeInit, and stalkInit ).

If User supplied file is selected, the specified MRC file will be used as the initial reference. In this case, the user is responsible for ensuring that the supplied reference is appropriately sized.  To support full searches, the reference should ideally be a cube large enough to contain a particle in any orientation, padded by twice the maximum search distance in any orientation.  (PEET will choose an adequate reference size automatically when the reference is not user-supplied).

Finally, if Multiparticle reference is selected the specified number (a power of 2 between 4 and 1024 chosen from the drop-down box) of randomly chosen particles will be aligned and combined using a binary tree structure, and the resulting average used as the initial reference.

2.6. Volume Size

Specifies the size in voxels of the subvolumes to be aligned and averaged.  Cubical volumes are prefered when possible, although they are not required.  NOTE: since PEET version 1.9.0 it is no longer necessary nor desirable that volume size be padded by twice the maximum Search Distance. Volume size should now be set only slightly larger than the volume of interest.

2.7. Missing Wedge Compensation Section

Check Enabled and specify Tilt Range in the Volume Table to use PEET's missing wedge compensation during alignment and 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.  Additionally, during alignment of particles against a reference with known missing wedge, correlations coefficients will be normalized by the fractional overlap between the informative regions of the particle and the reference. 

Edge shift 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 voxel is typical appropriate.

An additional type of missing wedge compensation is available when using cross-correlation to select particles for averaging at the end of each iteration (i.e. when creating a new reference or final averages).  If the number of Weight groups is set to a number larger than 1, particles are divided into groups based on their missing wedge orientation.  Correlation scores are then scaled so that each group has approximately the same median score.  The result is that nearly equal numbers of particles from each of the orientation groups will included in the reference (or the final average), thus minimizing a missing wedge bias. The appropriate setting must be chosen heuristically, but depends on the number of available particles as well as the number of distinct orientations present in the data.  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 in the X-Y plane), this type of compensation may not be necessary or helpful unless Random axial rotations (described below under Particle Y Axis Section) are used, or a program like modTwist2EM is used to provide varying starting orientations. Randomized particle selection, described below under , disables this type of missing wedge compensation.

Number of Tilt Axes determines how missing tomographic regions are specified. If 1 is selected, PEET will automatically generate single-tilt-axis missing wedges corresponding to the tilt ranges in the volume table. If 2 or more is selected, PEET will read binary missing wedge masks from the files whose names are specified under Missing Wedge Mask. For dual- or multi-tilt axis tomograms, programs dualAxisMask or multiTiltMask can be used to generate the necessary mask file(s).

2.8. Masking Section

Users can specify a mask to limit the portion of the reference volume to be included in cross-correlation calculations.  The default, None, is to use the entire volume.  Spherical, cylindrical, or user-defined masks can be selected by checking the appropriate radio button.  For both spherical and cylindrical masks, both inner and outer radii can be specified, allowing definition of either solid regions or hollow shells. Voxels between the inner and outer radii will be included. Truncated cylindrical masks can also be generated using the optional Cylinder Height field.

To use an MRC volume containing an arbitrary mask select the User supplied binary file option, and enter the name of a file containing the mask volume.  By default, masks are treated as if they were binary. I.e. A voxel of the reference volume will be masked out (i.e. excluded) if the value of its corresponding voxel in the mask volume is zero and included otherwise.  The mask volume size need not match that of the reference.  If they differ, the mask volume will be centered on the reference, and mask values outside the reference volume ignored.  Mask values outside the mask volume, but within the reference, will be 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 customized mask volume is to use IMOD program imodmop, which can generate arbitrary shapes. By placing closed contours on each section of interest, the region inside the contour will be included. When masks are treated as binary it does not matter that imodmop leaves original density values rather than 1's in the region that is retained.

Masks will be centered on the initial reference, and, by default, a cylindrical mask will be oriented with its long axis along the reference's Y axis.  You can override the orientation of a cylindrical mask by selecting Manual Cylinder Orientation and specifying the desired Z and Y rotations in degres.

By default, masks are treated as binary, leading to an abrupt edge. To generate a soft-edged mask, enter a positive value for Blur mask by, which specifies the standard deviation of a 3D Gaussian to be convolved with the original, binary mask before use. The resulting soft edge will extend approximately 2.5 standard deviations on either side of the original abrupt transition.

2.9. Particle Y Axis Section

The particle Y axis (or twist axis) is the axis PEET uses for the first angular search, referred to as Phi in the iteration table.  (Particle X and Z axes are determined from Y to form a right handed coordinate system, with some arbitrary conventions to resolve ambiguities).  if Tomogram Y axis is selected, the particle Y axis will coincide with the tomogram Y axis.  In this case, Phi will be around the tomogram Y axis, Theta around the tomogram Z axis, and Psi around the tomogram X axis.

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 lie along continuous contours, e.g. along a filament such as a microtubule.  When using this option, be sure to model each filament in a separate contour.

End points of contour is similar to Particle model points, except that the vector between the first and the last points in the contour will be taken as the Y axis for each point in that contour.  This can be useful when the contours are nearly straight, as it provides a less noisy estimate of the Y axis.

2.10. Initial Motive List Section

Controls in this section provide several options for setting up an initial motive list with starting orientations for all particles.  This can allow limiting the angular search range, improving both throughput and accuracy, even when the particles have significantly different starting orientations. 

Set all angles to zero specifies that 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 extract such information, preferring instead to rely on a more extensive alignment search.

Align particle Y axes will automatically generate initial motive list rotations to align each particle's Y axis with that of the reference. This is particularly useful when modeling filaments and Particle Y axis is set to Particle model points or End points of contour.

User supplied csv files uses rotations and shifts specified in user-supplied motive list(s). Such motive list (MOTL) files can be obtained from a prior PEET run or from auxiliary programs such as meshInit, modTwist2EM, slicer2MOTL, spikeInit, or stalkInit.

Uniform random rotations will select a random rotation drawn from a uniform distribution for each particle. In conjunction with a limited range angular search, this can be useful in avoiding missing wedge bias for isolated particles. With suspected icosahedral symmetry, for example, a useful approach is to use this option along with angular search ranges of roughly +/- 32 degrees. Note that random rotations will only help avoid missing wedge bias if other knowledge allows using less than a full spherical search.

Random axial (Y) rotations will align the particle Y axes to that of the reference, followed by a random rotations around that axis. This can be helpful in minimizing missing wedge bias, e.g. when averaging sections of a cylindrcal filament with similar missing wedge orientations and known axial symmetry (e.g. a 15 protofilament microtubule). Random axial rotations will help eliminate missing wedge bias only if other knowledge allows restricting the axial (Phi) search range.

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 Using Parallel Processing in the Etomo User's Guide for details on these controls and on configuring parallel processing. For information on using a cluster queue, see the IMOD man pages for processchunks and the cpu.adoc file. To use PEET on a Slurm cluster, please also see the 3 Slurm examples near the end of the sample $IMOD_DIR/autodoc/cpu.doc shipped with Imod.

3.2. Iteration Table

Particles per CPU controls the splitting of alignment searches into "chunks" for parallel execution on multiple cpus. No more than the specified number of particles will be sent simultaneously to a single cpu for alignment. The smaller this value, the more command and log files will be created, and, within limits, the greater the overall throughput. Too high a value, can lead to long delays should you wish to pause and then resume processing. The default of 20 is a good choice for most purposes (runs with at least several hundred subvolumes); if etomo reports more than a few thousand chunks to be processed, you may wish to raise this setting. Conversely, if you have many cpus and few particles, you can lower Particles per cpu for faster execution. To make effective use of available processors, the total number of chunks reported by etomo should be no less than the number of cpus used times the number of alignment iterations to be performed.

Following rows of the volume table each control a single iteration and have five or six types of entries.

Angular Search Range sets search ranges and increments (step sizes) in degrees for the 3 rotations (Phi, Theta, and Psi), referring to rotations around particle Y, Z, and X axes, respectively. For example, if Max and Step fields of Phi are set to 6 and 2 respectively, Phi will will assume values (-6, -4, -2, 0, 2, 4, and 6). A general guideline is that except for special cases (e.g. a no-search iteration to generate a new reference) earlier iterations should have larger ranges and step sizes than later ones.  Multiple iterations with larger step sizes are typically faster than a single search with a large range and small step size.  A reasonable compromise is to use a Max of 3 times Step, as in the example above.  Both Max and Step can then be reduced by a factor of 2 at each subsequent iteration, until the desired precision is achieved.  In many cases, it may be necessary to use more steps than this during the first iteration(s) to obtain a reasonable initial alignment.  To skip searching around a particular axis entirely, set the Max to 0 and Step to 1.

Search Distance 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 limits are in tomogram rather than particle coordinates, unless option Search along particle axes on the More Options tab is checked, so using different limits would be useful only if particles all have similar orientations with respect to at least one tomogram axis.  This entry typically has a minor effect on execution time, but small values can make the alignment more reliable by avoiding spurious correlation peaks at higher translations.  A search distance of 0 can be specified to disable searching, e.g. to verify the quality of the initial alignment.

Low-Pass Filter specifies parameters for filtering out high frequencies.  Frequencies are in reciprocal voxels, 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 cutoff, the response falls like the right half of a Gaussian with standard deviation given by the Sigma field.  By default, both the reference and the particle are filtered.  One way to see the effect of a given filter on a particular volume or particle is to apply that filter using IMOD program mtffilter, e.g., with the command "mtffilter -3d -low cutoff, sigma input_file output_file".  Alternatively, you can explore the effect on a selected image plane interactively by opening the volume in 3dmod choosing Edit-->Image-->Process-->Fourier filter from the menu.

High-Pass Filter will appear if Bandpass filtering is checked. Entries are Cutoff and Sigma, as for the Low-Pass Filter, but in this case the cutoff specifies the frequency below which there is no response. Above cutoff, the response increases like the left half of a Guassian with the specified standard deviation. Cutoff <= 0 disables high-pass filtering. High-pass filtration is seldom needed and typically used only when there is a gradient across the volumes which must be removed.

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 (unless Randomized particle selection is specified under The More Options Tab. 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, especially if limited data are available or at the first iteration to minimize 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. 

Duplicate Tolerance provides control over PEET's duplicate rejection logic.  Depending on the particle spacing and search distance, alignment 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 duplicate particles after each iteration is checked, 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.  Two particles are considered duplicates only if their separation exceeds neither of these tolerances. 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 ignored 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.

Normally PEET treats the Angular Search Ranges and Search Distance for each iteration as completely separate from those at any other iteration. So, for example, if Search Distances of 5, 4, and 3 voxels were specified at each of 3 iterations, it is possible that a particle could be translated by up to 5+4+3 = 12 voxels in each of the X, Y, and Z directions. If Strict search limit checking is checked, the maximum shift from the starting position in each direction is limited to the largest distance specified at any iteration... in this case 5 voxels. Maximum Angular Search Ranges are treated similarly.

As for the Volume Table, action buttons to the right of the Iteration Table allow adding, deleting, or rearranging lines in the table.

3.3.Spherical Sampling for Theta and Psi Section

This option makes full angular searching at the first iteration more efficient by sampling orientation space at relatively uniform intervals.  This 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.  Simply varying both Theta and Psi with regular increments results in oversampling near the poles.  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.  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 Step for Phi in the Iteration Table, as well as the Sample interval for spherical samping.  Typically Sample interval should be set equal to the Phi Step size.

3.4. Number of Particles to Average

Specify how many particles to use to form averages.  For example, if Start, Step, End, and Additional numbers are set to to 50, 50, 200, and 205, respectively, there will be 5 final averages, containing 50, 100, 150, 200 and 205 particles.  Values listed under Additional numbers must be comma separated, monotonically increasing, and greater than the End value of the arithmetic sequence.  .

3.5. Use Equal Numbers of Particles from All Tomograms

These checkboxes provide another tool for reducing bias when combining data from multiple tomograms.  If checked, PEET will attempt to use equal numbers of particles from each tomogram when computing averages or new references.  (Unequal numbers may still be used if required to achieve the requested number of particles).  If left unchecked, it will choose particles without regard to which tomogram(s) they are in.

3.6. Action Buttons

After reviewing and selecting Systems and / or processors in the Processing Table, press Run to start 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 requested computations. During processing, status will be indicated by progress bar at the top of the page. Additional status information may be obtained from the messages in the optionally visible log window, as well as from the Parallel processing section.  Note that once a run has been started it is permissible to close the etomo gui and even to log out, if desired; the run will continue. If the same project is later opened in a new etomo window on the same system from which it was originally run, it will reconnect to any processes still running and display the current status.

When processing is complete, press Open Averaged Volumes in 3dmod to view the computed averages in 3dmod.  The Zap and Isosurface windows will be opened automatically.  The isosurface threshold can be adjusted independently for each average. Similarly, pressing Open Reference Files in 3dmod will allow you to view the references generated at each iteration.  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 which do not affect the alignment.

4. The More Options Tab

This tab contains additional, optional settings. Please note that some, less-frequently used options, are not available via the etomo gui, and can only be accessed by manually editing the parameter file.

4.1. Particle Selection

Exclude particles and Include particles are lists of particles numbers (numbered consecutively from 1 in the 1st tomogram) to be excluded or included during averaging and related operations.

Similarly, Average only members of classes is a list of class numbers whose members should be included. This is most often useful after classification with programs pca and clusterPca, but can be used whenever numeric class labels are available in column 20 of the motive list.

Multiple classes or particles can be specified separated by commas, with ranges indicated with ":". Include particles is processed first, followed by Exclude particles, and, finally, Average only members of classes.

Elevation Compensation adjusts cross-correlation scores as a function of their elevation angle prior to particle selection for averaging. This can help equalize the frequency of polar versus equatorial views.

Randomized particle selection indicates that particles should be selected for inclusion in averages or new references at random, rather than based on their cross-correlation scores. Randomized particle selection is incompatible with Missing Wedge Compensation during alignment and will effectively set the number of Weight Groups to 1.

4.2. Alignment

Fast rotational matching allows use of fast rotational matching during rotation only alignment searches, when applicable.

Filter reference only specifies that any low pass or bandpass filtration will be applied to the reference only rather than to both the reference and individual particles.

Floating point wedge mask allows floating point (rather than binary) user-supplied wedge masks.

Masked correlation computation specifies the use of a slower compuation for cross-correlation which is potentially more accurate when masking is in use.

Search along particle axes specifies that search distances are to be applied as (approximately) along particle rather than tomogram axes.

If Use absolute value of cross-correlation is checked, PEET will maximize the absolute value of the cross-correlation, rather than its signed value, during alignment searches. This can help prevent noise from reinforcing to match features in the reference and works well for many biological structures which are globular or irregularly shaped. Use it with caution, however, with repeating or highly symmetric structures where there is a chance of aligning "out of phase" (i.e. dark on light).

Per iteration c<N> axial search order is a list specifying axial search order for each alignment iteration. If the ith entry is n (> 1), cN symmetry will be applied to the both the reference and particles ference before performing the ith iteration alignment search, which will be in Phi only from -180/n to 180/n degrees in steps of dPhi.

4.3. Processing

If Align averages to have their Y axes vertical is selected, average volumes will be reoriented to have their Y axis approximately vertical. This is particularly useful for particles along straight or slightly curved filaments. Note that the reorientation is applied only to the particles as they are averaged and will not be reflected in the standard motive lists. (Special motive lists with Vertical in their name(s) which do include thes rotations will also be written).

No reference refinement allows for alignment against a fixed reference (i.e. template matching). Normally, PEET generates a new reference at the end of each iteration based on the current alignment. If no reference refinement is checked, the initial reference (whether user supplied our automatically generated), will be used at all iterations without modification.

c<N> symmetric averaging with N=? specifies imposition of cN symmetry during all averaging operations including construction of new references. This assumes the cN symmetry axis has been centered and oriented along Y, and imposes symmetry more strictly than prior symmetry expansion. This also allows faster alignment using the smaller, non-expanded set of particles.

If Use previously extracted particles is checked, previously extracted particles will be used rather than automatically extracting particles on the fly. Input "volumes" must be stacks of extracted particles rather than full tomograms in this case. Please see the extractAllParticles, extractParticles, and PEET man pages for details.

If Save individual aligned particles is checked, each individual particle contributing to one of the final averages will be saved to an MRC file named aligned*.mrc. This can consume large amounts of disk space.

Masking during c<N> averaging allows use of a soft spherical mask during c<N> symmetric averaging.

Debug level (0, 1, 2, or 3) controls the amount of information in the log filess, with higher numbers leading to more verbose logs.

5. Resampling Existing Volumes / Models

In some cases it may be desirable to change the origin, image size, or voxel size of an existing IMOD volume and its corresponding model for use with PEET.  For example, one may wish to create a binned version to reduce memory requirements or to speed preliminary characterization.  This is easy using IMOD, but must be done in a specific way to preserve compatability with both PEET and 3dmod.

First, it is necessary to ensure the starting model contains information about the associated volume.  To do this, simply open the volume and the model together in 3dmod, and then re-save the model;  equivalently, use "imodtrans -i" to update the existing model, as illustrated below.  Second, create the modified volume using IMOD tools such as "binvol" or "clip -resize". Finally, generate the revised model either by a) opening the modified volume and the original model together in 3dmod, or b) by using "imodtrans -i".

For example, to perform 2-fold binning on a dataset in which the original volume and model were named "old.rec" and "old.mod", respectively, one might use

imodtrans -i old.rec old.mod old.mod
binvol -b 2 old.rec new.rec
imodtrans -i new.rec old.mod new.mod
In this case, the first operation could also have been done by running "3dmod old.rec old.mod" and then selecting the "File / Save" menu entry.  Similarly, the final operation could have been done by running "3dmod new.rec old.mod", selecting "File / Save As", and specifying "new.mod" as the name for the new model.

If the voxel size has been changed, parameter file settings (e.g. mask radii) must be modified accordingly.  Similarly, any non-zero offsets in columns 11-13 of the motive list(s) will need to be scaled manually; motive lists are stored in comma-separated-value (csv) format, so a spreadsheet program can be used to make any changes required. Alternatively, scaling can be avoided by using createAlignedModel to generate new models and motive list(s) with no offsets.