Segrefine3dgrid¶

Program to optimize segmentrefine3d reconstruction by varying refinement parameters systematically on a grid

Input: Image input stack refinement

Output: Output volume name

Parameters¶

Parameter	Example (default)	Description
Image input stack refinement	protein_stack.hdf	Input stack should have CTF applied and prepared: accepted image file formats (hdf).
Output volume name	recvol.hdf	Output name for volumes of iterative structure refinement (completion to ‘prefix_XXX.ext’): accepted image file formats (hdf).
First parameter	helical_rise_or_pitch	Choose parameter to be varied in first dimension: ; ‘number_of_iterations’; ‘high_pass_filter_cutoff’; ‘low_pass_filter_cutoff’; ‘b-factor’; ‘include_classes’; ‘exclude_curvature_above’; ‘exclude_layer_line_ccc_below’; ‘exclude_defocus_below’; ‘exclude_defocus_above’; ‘exclude_astigmatism_above’; ‘exclude_projection_ccc_below’; ‘exclude_out_of_plane_tilt_below’; ‘exclude_out_of_plane_tilt_above’; ‘exclude_shift_normal_to_helix_above’; ‘helical_rise_or_pitch’; ‘helical_rotation_or_number_of_units_per_turn’; ‘helix_start’; ‘delta_in_plane_rotation’; ‘out_of_plane_tilt_search_range’; ‘out_of_plane_count’; ‘azimuthal_count’; ‘alignment_image_size’; ‘segmentation_step’; ‘pixelsize’;
Second parameter	helical_rotation_or_number_of_units_per_turn	Choose parameter to be varied in second dimension: ; ‘none’; ‘number_of_iterations’; ‘high_pass_filter_cutoff’; ‘low_pass_filter_cutoff’; ‘b-factor’; ‘include_classes’; ‘exclude_curvature_above’; ‘exclude_layer_line_ccc_below’; ‘exclude_defocus_below’; ‘exclude_defocus_above’; ‘exclude_astigmatism_above’; ‘exclude_projection_ccc_below’; ‘exclude_out_of_plane_tilt_below’; ‘exclude_out_of_plane_tilt_above’; ‘exclude_shift_normal_to_helix_above’; ‘helical_rise_or_pitch’; ‘helical_rotation_or_number_of_units_per_turn’; ‘helix_start’; ‘delta_in_plane_rotation’; ‘out_of_plane_tilt_search_range’; ‘out_of_plane_count’; ‘azimuthal_count’; ‘alignment_image_size’; ‘segmentation_step’; ‘pixelsize’;
Lower and upper limit first parameter	(1.4, 1.9)	Lower and upper limit of first parameter for grid search. Unit dependent on quantity (accepted values min=-1e+07, max=1e+07).
Lower and upper limit second parameter	(22.0, 24.0)	Lower and upper limit of second parameter for grid search. Unit dependent on quantity (accepted values min=-1e+07, max=1e+07).
First and second parameter increment	(0.1, 0.3)	First and second parameter increment for grid search. Unit dependent on quantity (accepted values min=-1e+08, max=1e+08).
Diagnostic plot prefix	diagnostic_plot.pdf	Output name for diagnostic plots of iterative structure refinement (completion to ‘prefix_XXX.ext’): accepted file formats (pdf, .png, .bmp, .emf, .eps, .gif, .jpeg, .jpg, .ps, .raw, .rgba, .svg, .svgz, .tif, .tiff).
spring.db file	spring.db	Program requires a combined spring.db from segment (and optional: micctfdetermine). An updated spring.db will be created in the working directory.
Estimated helix inner and outer diameter in Angstrom	(0, 190)	Generous inner and outer diameter of helix required for cylindrical mask in Angstrom (accepted values min=0, max=1500).
Pixel size in Angstrom	1.163	Pixel size is an imaging parameter (accepted values min=0.001, max=100).
Symmetrize helix	True	Tick to impose helical symmetry. Symmetry-related views are incorporated into final reconstruction.
Helical rise/rotation or pitch/number of units per turn choice	rise/rotation	Choose whether helical ‘rise/rotation’ or ‘pitch/unit_number’ of units per turn pairs are given for generating the helical lattice.
Helical symmetry in Angstrom or degrees	(1.408, 22.03)	Helical rise/pitch (Angstrom) and rotation (degrees)/number of units per turn to be imposed to 3D reconstruction (accepted values min=-1000, max=1000).
Image alignment size in Angstrom	700	Image dimension to be used for alignment in Angstrom, i.e. the larger, the more signal is available for alignment but less compensation of structural flexibility is possible (accepted values min=10, max=5000).
Step size of segmentation in Angstrom	70	Overlapping segments are related views according to helical symmetry, i.e. step size should be a multiple of helical rise (stepsize of 0 corresponds to one central box per helix) (accepted values min=0, max=2000).

Sample parameter file¶

You may run the program in the command line by providing the parameters via a text file:

segrefine3dgrid --f parameterfile.txt

Where the format of the parameters is:

Image input stack refinement             = protein_stack.hdf
Output volume name                       = recvol.hdf
First parameter                          = helical_rise_or_pitch
Second parameter                         = helical_rotation_or_number_of_units_per_turn
Lower and upper limit first parameter    = (1.4, 1.9)
Lower and upper limit second parameter   = (22.0, 24.0)
First and second parameter increment     = (0.1, 0.3)
Diagnostic plot prefix                   = diagnostic_plot.pdf
spring.db file                           = spring.db
Estimated helix inner and outer diameter in Angstrom = (0, 190)
Pixel size in Angstrom                   = 1.163
Symmetrize helix                         = True
Helical rise/rotation or pitch/number of units per turn choice = rise/rotation
Helical symmetry in Angstrom or degrees  = (1.408, 22.03)
Image alignment size in Angstrom         = 700
Step size of segmentation in Angstrom    = 70

Additional parameters (intermediate level)¶

Parameter	Example (default)	Description
Number of iterations	20	Number of iteration cycles of projection, alignment and 3D reconstruction (accepted values min=1, max=200).
Reference structure option	False	Tick if reference available - otherwise uses cylinder with diameter specifications below.
Reference volume	reference_vol.hdf	Reference to be used for 3D structure refinement: accepted image file formats (hdf).
Continue refinement option	False	To continue previous refinement and use final alignment parameters as starting point. In case no reference structure is provided, it will re-create the final 3D reconstruction without the projection matching step. When a reference structure is provided, it will perform projection matching and 3D reconstruction.
refinement.db file	refinement.db	Program requires a refinement.db from previous segmentrefine3d. An updated refinement.db will be created in the working directory.
Assemble refinement strategy	True	Assemble refinement strategy according to resolution aims. If not ticked it will perform refinement from low to maximum resolution. For a lower resolution aim, data will be binned and refinement consequently converges and refines quicker. Subsequently, such refinements can be used as a starting point for later higher-resolution reconstructions.
LR - Low resolution aim	True	Select if your resolution aim is ‘low’(>20 Angstrom). Data will be binned internally to speed up refinement. Volumes are symmetrized in 3D.
MR - Medium resolution aim	True	Select if your resolution aim is ‘medium’(10 - 20 Angstrom). Data will be binned to maximum spatial resolution of 10 Angstrom. Volumes are symmetrized in 3D.
HR - High resolution aim	True	Select if your resolution aim is ‘high’(5 - 10 Angstrom). Data will be binned internally to maximum spatial resolution of 5 Angstrom.
MaxR - Maximum resolution aim	True	Select if your resolution aim is your maximum spatial frequency. Data will not be binned and is limited by pixelsize. As a result, this refinement is very slow.
Absolute X and Y translation limit in Angstrom	(100, 100)	Absolute translation limit of alignment search in Angstrom perpendicular and along helix axis from center of segment. This is useful because iterative alignments tend to move the helix axis in y-direction (accepted values min=1, max=1000).
Independent half-set refinement	False	Independent half-set refinement from two halves of the data set that are .kept separate for multiple cycles. Sometimes referred to ‘gold standard refinement’. Here, helices are divided into odd and even data sets.
Half-set refinement start	medium	Choose when to start independent half-set refinement. At least, a low resolution refinement is critical to compute reliable FSC curves without an additional alignment step.
High-pass filter option	False	Option to high-pass filter images before alignment or band-pass filter in combination with low-pass filter.
Low-pass filter option	True	Option to low-pass filter images before alignment or band-pass filter in combination with high-pass filter.
High and low-pass filter cutoffs in 1/Angstrom	(0.001, 0.09)	Filter design for high- and low-pass filter with cutoffs in 1/Angstrom. Maximum spatial frequency is 1/(2*pixelsize) (accepted values min=0, max=1).
Keep intermediate files	False	Keep intermediate projection and diagnostic images (power spectra and reprojections) which are iteratively generated - EM image stacks are deleted otherwise.
Rotational symmetry	1	Additional x-fold rotational symmetry or x-number of helix start (accepted values min=1, max=100).
Helix polarity	polar	Choose whether helix is ‘polar’ or ‘apolar’. Polar helices have different ends at the top and bottom. Only the predominant direction within a helix will be used for the reconstruction. In apolar helices they are related by 180 degree rotation. Thus each segment can be inserted twice in the 3D reconstruction in opposite directions.
Force helical continuity	True	Tick to enforce helical continuity of segments. This way segments are removed when polarity flips within helix. Running averages of in-plane rotation, shifts perpendicular to helix axis and out-of-plane angles are computed and monitored. Forward differences and polarity distribution are given in statistical evaluation.
Limit in-plane rotation	True	Restricts in-plane rotation alignment.
Delta in-plane rotation angle	10.0	Rotational alignment is being performed within +/- delta angle from 0 and 180 degrees (delta=0 means only 0/180 are checked, delta=180 means no rotational restraint) (accepted values min=0, max=180).
Out-of-plane tilt angle range	(-12, 12)	Expected out-of-plane tilt angle in degrees (0=no out-of-plane tilt) of helices used for 3D reconstruction (accepted values min=-40, max=40).
Number of projections azimuthal/out-of-plane angle	(90, 7)	Number of projections (e.g. 90 azimuthal projections per 360 degrees - projection every four degrees, out-of-plane tilt 7 projections from -12 to 12 degrees to be searched for 3D reconstruction. High- and maximum resolution reconstructions will be further refined by 5 * number of specified azimuthal and out-of-plane angles (accepted values min=1, max=1000).
MPI option	True	OpenMPI installed (mpirun).
Number of CPUs	8	Number of processors to be used (accepted values min=1, max=1000).
Temporary directory	/tmp	Temporary directory should have fast read and write access.

Sample parameter file (intermediate level)¶

You may run the program in the command line by providing the parameters via a text file:

segrefine3dgrid --f parameterfile.txt

Where the format of the parameters is:

Image input stack refinement             = protein_stack.hdf
Output volume name                       = recvol.hdf
First parameter                          = helical_rise_or_pitch
Second parameter                         = helical_rotation_or_number_of_units_per_turn
Lower and upper limit first parameter    = (1.4, 1.9)
Lower and upper limit second parameter   = (22.0, 24.0)
First and second parameter increment     = (0.1, 0.3)
Diagnostic plot prefix                   = diagnostic_plot.pdf
Number of iterations                     = 20
Reference structure option               = False
Reference volume                         = reference_vol.hdf
spring.db file                           = spring.db
Continue refinement option               = False
refinement.db file                       = refinement.db
Assemble refinement strategy             = True
LR - Low resolution aim                  = True
MR - Medium resolution aim               = True
HR - High resolution aim                 = True
MaxR - Maximum resolution aim            = True
Absolute X and Y translation limit in Angstrom = (100, 100)
Independent half-set refinement          = False
Half-set refinement start                = medium
High-pass filter option                  = False
Low-pass filter option                   = True
High and low-pass filter cutoffs in 1/Angstrom = (0.001, 0.09)
Keep intermediate files                  = False
Estimated helix inner and outer diameter in Angstrom = (0, 190)
Pixel size in Angstrom                   = 1.163
Symmetrize helix                         = True
Helical rise/rotation or pitch/number of units per turn choice = rise/rotation
Helical symmetry in Angstrom or degrees  = (1.408, 22.03)
Rotational symmetry                      = 1
Helix polarity                           = polar
Force helical continuity                 = True
Limit in-plane rotation                  = True
Delta in-plane rotation angle            = 10.0
Out-of-plane tilt angle range            = (-12, 12)
Number of projections azimuthal/out-of-plane angle = (90, 7)
Image alignment size in Angstrom         = 700
Step size of segmentation in Angstrom    = 70
MPI option                               = True
Number of CPUs                           = 8
Temporary directory                      = /tmp

Additional parameters (expert level)¶

Parameter	Example (default)	Description
Subgrid option	False	Run subgrids to parallelize expensive grid searches.
Part and number of subgrids	(1, 3)	E.g. one out of total of three subgrids will be run. This features is thought for parallelization of expensive grid searches (accepted values min=1, max=100).
Grid continue option	False	Continue grid refinement in case of interrupted grid searches.
Grid database	grid.db	Continue grid refinement in case of interrupted grid searches.
LR - azimuthal and out-of-plane search restraint in degrees	(180.0, 180.0)	Restrain low-resolution projection matching search of azimuthal and out-of-plane angles within specified limits of previous angle. 0 degrees = fixed, i.e. only previous projection will be searched. 360 degrees = no restraints, i.e. all projections are searched (accepted values min=0, max=180).
LR - X and Y translation range in Angstrom	(50, 23)	Low resolution translational range of alignment search perpendicular and along helix axis (X: off-center helical axis, Y: (minimum y-range=helical rise/2) (accepted values min=0, max=1000).
MR - azimuthal and out-of-plane search restraint in degrees	(180.0, 180.0)	Restrain medium-resolution projection matching search of azimuthal and out-of-plane angles within specified limits of previous angle. 0 degrees = fixed, i.e. only previous projection will be searched. 360 degrees = no restraints, i.e. all projections are searched (accepted values min=0, max=180).
MR - X and Y translation range in Angstrom	(21, 10)	Medium resolution translational range of alignment search perpendicular and along helix axis (X: off-center helical axis, Y: (minimum y-range=helical rise/2) (accepted values min=0, max=1000).
HR - azimuthal and out-of-plane search restraint in degrees	(20.0, 20.0)	Restrain high-resolution projection matching search of azimuthal and out-of-plane angles within specified limits of previous angle. 0 degrees = fixed, i.e. only previous projection will be searched. 360 degrees = no restraints, i.e. all projections are searched (accepted values min=0, max=180).
HR - X and Y translation range in Angstrom	(14, 7)	High resolution translational range of alignment search perpendicular and along helix axis (X: off-center helical axis, Y: (minimum y-range=helical rise/2) (accepted values min=0, max=1000).
MaxR - azimuthal and out-of-plane search restraint in degrees	(2.0, 2.0)	Restrain maximum-resolution projection matching search of azimuthal and out-of-plane angles within specified limits of previous angle. 0 degrees = fixed, i.e. only previous projection will be searched. 360 degrees = no restraints, i.e. all projections are searched (accepted values min=0, max=180).
MaxR - X and Y translation range in Angstrom	(7, 3.5)	Maximum resolution translational range of alignment search perpendicular and along helix axis (X: off-center helical axis, Y: (minimum y-range=helical rise/2) (accepted values min=0, max=1000).
Frame motion correction	False	Segment-based motion correction to correct for beam-induced movement.
Frame average window size	3	Size of window of number of frames used for running average frame processing. 1 means no additional frame averaging, 3 corresponds to +/-1 average. Only odd values will be considered. Even values will be rounded to the next higher odd one, e.g. 4 to 5 (accepted values min=0, max=1000).
Frame local averaging distance	700	Distance in Angstrom used for local average frame processing. Movement of segments between frames will be averaged over neighboring segments of this distance within helix. 0 means no local averaging. (accepted values min=0, max=10000).
B-Factor	0	B-Factor in 1/Angstrom^2 to be applied to images. Zero no modulation of frequencies. Negative B-factors enhance high-resolution frequencies. Positive B-factors dampen high-resolution frequencies (accepted values min=-50000, max=50000).
Custom filter option	False	Option to custom filter images before alignment.
Custom-built filter file	filter_function.dat	Custom-built filter function with two columns (normalized spatial frequency 0 - 0.5, Fourier filter coefficients between 0 - 1): accepted file formats (txt, .dat).
Automatic FSC filter	True	Automatic filter design derived from square root of Fourier Shell Correlation (FSC) between reconstructions from half data sets.
Micrographs select option	False	Choose whether to select any particular micrographs.
Include or exclude micrographs	include	Choose whether to ‘include’ or ‘exclude’ specified micrographs.
Micrographs list	1-9, 11, 13	List of comma-separated micrograph ids, e.g. ‘1-10, 12, 14’ (1st micrograph is 1).
Helices select option	False	Choose whether to select any particular helices.
Include or exclude helices	include	Choose whether to ‘include’ or ‘exclude’ specified helices.
Helices list	1-9, 11, 13	List of comma-separated helix ids, e.g. ‘1-10, 12, 14’ (1st helix is 1).
Segments select option	False	Choose whether to select any particular segments.
Include or exclude segments	include	Choose whether to ‘include’ or ‘exclude’ specified segments.
Segment file	stackid_file.dat	File with single column of stack_ids.
Classes select option	False	Choose whether to select any particular classes.
Include or exclude classes	include	Choose whether to ‘include’ or ‘exclude’ specified classes.
Class type	class_id	Choose class type either ‘class_id’ based on segmentclass assignments or ‘class_model_id’ based on segmultirefine3d/segclassmodel assigments.
Classes list	1-9, 11, 13	List of comma-separated class ids, e.g. ‘1-10, 12, 14’ (1st class is 0).
Persistence class option	False	Choose whether to select segments from classes based on class assignments of neighboring segments.
Persistence class length in Angstrom	700	Length of helix window that will be used to average the class memberships of segments (accepted values min=1, max=5000).
Class occupancy threshold	0.5	Class occupancy threshold of segments (between 0 and 1) that belong to specified subset of classes within persistence length. Segments that have a lower occupancy will be excluded (accepted values min=0, max=1).
Straightness select option	False	Choose whether to select any helices based on straightness.
Include or exclude straight helices	include	Choose whether to ‘include’ or ‘exclude’ helices of specified persistence length.
Persistence length range	(80, 100)	Range of persistence length in percent, i.e. upper 10 percent of distribution is expressed as 90 - 100 percent range, lower 20 percent is expressed as 0 - 20 percent etc. 90 - 100 % corresponds to most straight helices. Values from database are stored in m, e.g. ‘0-0.0001’ Persistence length is calculated as: p = -ln(2 * (end_to_end_distance / contour_length) ** 2 - 1) / contour_length)), i.e. short persistence lengths of 1 nm correspond to very flexible whereas 1 m corresponds to extremely straight helices. Examples are TMV: 2.9 mm (2.9e-3 m), amyloid beta filaments: 300 microm (3e-4 m) and DNA: 100 nm (1e-7 m). Due to the alignment error of the segments this value may not be absolutely comparable to determined persistence lengths by other methods but still be valid as a relative measure of straightness (accepted values min=0, max=100).
Layer line correlation select option	False	Choose whether to select any segments based on layer-line cross-correlation coefficient.
Include or exclude segments based on layer-line correlation	include	Choose whether to ‘include’ or ‘exclude’ segments of specified cross correlation coefficient with layer lines.
Correlation layer line range	(60, 100)	Range of cross-correlation between layer lines of power spectrum average and segment in percent. i.e. upper 10 percent of distribution is expressed as 90 - 100 percent range, lower 20 percent is expressed as 0 - 20 percent etc. Values in database are stored as cross correlation coefficient, e.g. ‘0.5 - 1.0’ (accepted values min=0, max=100).
Defocus select option	False	Choose whether to select any segments based on defocus.
Include or exclude defocus range	include	Choose whether to ‘include’ or ‘exclude’ segments of specified defocus.
Defocus range	(10000, 40000)	Range of defocus in Angstrom, e.g. ‘10000-40000’ (accepted values min=0, max=100000).
Astigmatism select option	False	Choose whether to select any segments based on astigmatism.
Include or exclude astigmatic segments	include	Choose whether to ‘include’ or ‘exclude’ segments of specified astigmatism amplitude in Angstrom.
Astigmatism range	(0, 4000)	Range of astigmatism amplitude (difference between defocus one and two) in Angstrom, e.g. ‘0-4000’ (accepted values min=0, max=100000).
Projection correlation select option	False	Choose whether to select any segments based on matched cross-correlation coefficient.
Include or exclude segments based on projection correlation	include	Choose whether to ‘include’ or ‘exclude’ segments of specified cross correlation coefficient with projection.
Correlation projection range	(60, 100)	Range of cross-correlation peak between matched projection and segment in percent, i.e. upper 10 percent of distribution is expressed as 90 - 100 percent range, lower 20 percent is expressed as 0 - 20 percent etc. Values are stored as peak values depending on the pixel dimension, e.g. 1220 (accepted values min=0, max=100).
Out-of-plane tilt select option	False	Choose whether to select any segments based on out-of-plane tilt angle.
Include or exclude out-of-plane tilted segments	include	Choose whether to ‘include’ or ‘exclude’ segments of specified out-of-plane tilt angle.
Out-of-plane tilt range	(-5, 5)	Range of out-of-plane tilt in degrees, e.g. ‘-10 - 10 degrees’ (accepted values min=-50, max=50).
Shift normal to helix select option	False	Choose whether to select any segments based on forward shift difference normal to helix. This parameter tends to correlate with resolution.
Include or exclude segments with shift normal to helix	include	Choose whether to ‘include’ or ‘exclude’ segments of specified foward shift difference normal to helix. ‘include’ and 5 Angstrom will select for segments with a smaller forward difference. ‘exclude’ and 5 Angstrom will select for segments with a larger forward difference than 5 Angstrom.
Shift normal to helix in Angstrom	5.0	Shift normal to helix in Angstrom, e.g. ‘5’ (accepted values min=0, max=50).
Choose out-of-plane tilt amplitude correlation	False	Tick to manually to choose out-of-plane tilt range for computing sum of power spectra. This operation is relevant if you have very few segments or an unexpected out-of-plane tilt distribution. Otherwise no need to change it.
Amplitude correlation out-of-plane tilt range	(-12, 12)	Expected out-of-plane tilt angle in degrees (0=no out-of-plane tilt) of helices used for 3D reconstruction (accepted values min=-40, max=40).
3D CTF correction	True	Tick to perform 3D CTF correction by dividing through the average of all CTFs squared. Only applied in case segments were convolved by CTF. Therefore, if segments were phase-flipped no 3D CTF correction will be performed.
3D CTF correction intensity	low	Wiener filter constant as percent of Fourier amplitude. None - No 3D CTF correction. High - 40, 20, 10, 5. Medium - 20, 10, 5, 2. Low - 10, 5, 2, 1. (percent for low-, medium-, high- and maximum resolution refinement).

Sample parameter file (expert level)¶

You may run the program in the command line by providing the parameters via a text file:

segrefine3dgrid --f parameterfile.txt

Where the format of the parameters is:

Image input stack refinement             = protein_stack.hdf
Output volume name                       = recvol.hdf
First parameter                          = helical_rise_or_pitch
Second parameter                         = helical_rotation_or_number_of_units_per_turn
Lower and upper limit first parameter    = (1.4, 1.9)
Lower and upper limit second parameter   = (22.0, 24.0)
First and second parameter increment     = (0.1, 0.3)
Subgrid option                           = False
Part and number of subgrids              = (1, 3)
Grid continue option                     = False
Grid database                            = grid.db
Diagnostic plot prefix                   = diagnostic_plot.pdf
Number of iterations                     = 20
Reference structure option               = False
Reference volume                         = reference_vol.hdf
spring.db file                           = spring.db
Continue refinement option               = False
refinement.db file                       = refinement.db
Assemble refinement strategy             = True
LR - Low resolution aim                  = True
LR - azimuthal and out-of-plane search restraint in degrees = (180.0, 180.0)
LR - X and Y translation range in Angstrom = (50, 23)
MR - Medium resolution aim               = True
MR - azimuthal and out-of-plane search restraint in degrees = (180.0, 180.0)
MR - X and Y translation range in Angstrom = (21, 10)
HR - High resolution aim                 = True
HR - azimuthal and out-of-plane search restraint in degrees = (20.0, 20.0)
HR - X and Y translation range in Angstrom = (14, 7)
MaxR - Maximum resolution aim            = True
MaxR - azimuthal and out-of-plane search restraint in degrees = (2.0, 2.0)
MaxR - X and Y translation range in Angstrom = (7, 3.5)
Absolute X and Y translation limit in Angstrom = (100, 100)
Frame motion correction                  = False
Frame average window size                = 3
Frame local averaging distance           = 700
Independent half-set refinement          = False
Half-set refinement start                = medium
High-pass filter option                  = False
Low-pass filter option                   = True
High and low-pass filter cutoffs in 1/Angstrom = (0.001, 0.09)
B-Factor                                 = 0
Custom filter option                     = False
Custom-built filter file                 = filter_function.dat
Automatic FSC filter                     = True
Micrographs select option                = False
Include or exclude micrographs           = include
Micrographs list                         = 1-9, 11, 13
Helices select option                    = False
Include or exclude helices               = include
Helices list                             = 1-9, 11, 13
Segments select option                   = False
Include or exclude segments              = include
Segment file                             = stackid_file.dat
Classes select option                    = False
Include or exclude classes               = include
Class type                               = class_id
Classes list                             = 1-9, 11, 13
Persistence class option                 = False
Persistence class length in Angstrom     = 700
Class occupancy threshold                = 0.5
Straightness select option               = False
Include or exclude straight helices      = include
Persistence length range                 = (80, 100)
Layer line correlation select option     = False
Include or exclude segments based on layer-line correlation = include
Correlation layer line range             = (60, 100)
Defocus select option                    = False
Include or exclude defocus range         = include
Defocus range                            = (10000, 40000)
Astigmatism select option                = False
Include or exclude astigmatic segments   = include
Astigmatism range                        = (0, 4000)
Projection correlation select option     = False
Include or exclude segments based on projection correlation = include
Correlation projection range             = (60, 100)
Out-of-plane tilt select option          = False
Include or exclude out-of-plane tilted segments = include
Out-of-plane tilt range                  = (-5, 5)
Shift normal to helix select option      = False
Include or exclude segments with shift normal to helix = include
Shift normal to helix in Angstrom        = 5.0
Keep intermediate files                  = False
Estimated helix inner and outer diameter in Angstrom = (0, 190)
Pixel size in Angstrom                   = 1.163
Symmetrize helix                         = True
Helical rise/rotation or pitch/number of units per turn choice = rise/rotation
Helical symmetry in Angstrom or degrees  = (1.408, 22.03)
Rotational symmetry                      = 1
Helix polarity                           = polar
Force helical continuity                 = True
Limit in-plane rotation                  = True
Delta in-plane rotation angle            = 10.0
Out-of-plane tilt angle range            = (-12, 12)
Number of projections azimuthal/out-of-plane angle = (90, 7)
Image alignment size in Angstrom         = 700
Step size of segmentation in Angstrom    = 70
Choose out-of-plane tilt amplitude correlation = False
Amplitude correlation out-of-plane tilt range = (-12, 12)
3D CTF correction                        = True
3D CTF correction intensity              = low
MPI option                               = True
Number of CPUs                           = 8
Temporary directory                      = /tmp

Command line options¶

When invoking segrefine3dgrid, you may specify any of these options:

usage: segrefine3dgrid [-h] [--g] [--p] [--f FILENAME] [--c] [--l LOGFILENAME] [--d DIRECTORY_NAME] [--version] [--subgrid_option]
                       [--grid_continue_option] [--reference_structure_option] [--continue_refinement_option] [--assemble_refinement_strategy]
                       [--lr_-_low_resolution_aim] [--mr_-_medium_resolution_aim] [--hr_-_high_resolution_aim] [--maxr_-_maximum_resolution_aim]
                       [--frame_motion_correction] [--independent_half-set_refinement] [--high-pass_filter_option] [--low-pass_filter_option]
                       [--custom_filter_option] [--automatic_fsc_filter] [--micrographs_select_option] [--helices_select_option]
                       [--segments_select_option] [--classes_select_option] [--persistence_class_option] [--straightness_select_option]
                       [--layer_line_correlation_select_option] [--defocus_select_option] [--astigmatism_select_option]
                       [--projection_correlation_select_option] [--out-of-plane_tilt_select_option] [--shift_normal_to_helix_select_option]
                       [--keep_intermediate_files] [--symmetrize_helix] [--force_helical_continuity] [--limit_in-plane_rotation]
                       [--choose_out-of-plane_tilt_amplitude_correlation] [--3d_ctf_correction] [--mpi_option]
                       [input_output [input_output ...]]

Program to optimize segmentrefine3d reconstruction by varying refinement parameters systematically on a grid

positional arguments:
  input_output          Input and output files

optional arguments:
  -h, --help            show this help message and exit
  --g, --GUI            GUI option: read input parameters from GUI
  --p, --promptuser     Prompt user option: read input parameters from prompt
  --f FILENAME, --parameterfile FILENAME
                        File option: read input parameters from FILENAME
  --c, --cmd            Command line parameter option: read only boolean input parameters from command line and all other parameters will be assigned
                        from other sources
  --l LOGFILENAME, --logfile LOGFILENAME
                        Output logfile name as specified
  --d DIRECTORY_NAME, --directory DIRECTORY_NAME
                        Output directory name as specified
  --version             show program's version number and exit
  --subgrid_option, --sub
                        Run subgrids to parallelize expensive grid searches. (default: False)
  --grid_continue_option, --gri
                        Continue grid refinement in case of interrupted grid searches. (default: False)
  --reference_structure_option, --ref
                        Tick if reference available - otherwise uses cylinder with diameter specifications below. (default: False)
  --continue_refinement_option, --con
                        To continue previous refinement and use final alignment parameters as starting point. In case no reference structure is
                        provided, it will re-create the final 3D reconstruction without the projection matching step. When a reference structure is
                        provided, it will perform projection matching and 3D reconstruction. (default: False)
  --assemble_refinement_strategy, --ass
                        Assemble refinement strategy according to resolution aims. If not ticked it will perform refinement from low to maximum
                        resolution. For a lower resolution aim, data will be binned and refinement consequently converges and refines quicker.
                        Subsequently, such refinements can be used as a starting point for later higher-resolution reconstructions. (default: False)
  --lr_-_low_resolution_aim, --lr
                        Select if your resolution aim is 'low'(>20 Angstrom). Data will be binned internally to speed up refinement. Volumes are
                        symmetrized in 3D. (default: False)
  --mr_-_medium_resolution_aim, --mr
                        Select if your resolution aim is 'medium'(10 - 20 Angstrom). Data will be binned to maximum spatial resolution of 10 Angstrom.
                        Volumes are symmetrized in 3D. (default: False)
  --hr_-_high_resolution_aim, --hr
                        Select if your resolution aim is 'high'(5 - 10 Angstrom). Data will be binned internally to maximum spatial resolution of 5
                        Angstrom. (default: False)
  --maxr_-_maximum_resolution_aim, --max
                        Select if your resolution aim is your maximum spatial frequency. Data will not be binned and is limited by pixelsize. As a
                        result, this refinement is very slow. (default: False)
  --frame_motion_correction, --fra
                        Segment-based motion correction to correct for beam-induced movement. (default: False)
  --independent_half-set_refinement, --ind
                        Independent half-set refinement from two halves of the data set that are .kept separate for multiple cycles. Sometimes
                        referred to 'gold standard refinement'. Here, helices are divided into odd and even data sets. (default: False)
  --high-pass_filter_option, --hig
                        Option to high-pass filter images before alignment or band-pass filter in combination with low-pass filter. (default: False)
  --low-pass_filter_option, --low
                        Option to low-pass filter images before alignment or band-pass filter in combination with high-pass filter. (default: False)
  --custom_filter_option, --cus
                        Option to custom filter images before alignment. (default: False)
  --automatic_fsc_filter, --aut
                        Automatic filter design derived from square root of Fourier Shell Correlation (FSC) between reconstructions from half data
                        sets. (default: False)
  --micrographs_select_option, --mic
                        Choose whether to select any particular micrographs. (default: False)
  --helices_select_option, --hel
                        Choose whether to select any particular helices. (default: False)
  --segments_select_option, --seg
                        Choose whether to select any particular segments. (default: False)
  --classes_select_option, --cla
                        Choose whether to select any particular classes. (default: False)
  --persistence_class_option, --per
                        Choose whether to select segments from classes based on class assignments of neighboring segments. (default: False)
  --straightness_select_option, --str
                        Choose whether to select any helices based on straightness. (default: False)
  --layer_line_correlation_select_option, --lay
                        Choose whether to select any segments based on layer-line cross-correlation coefficient. (default: False)
  --defocus_select_option, --def
                        Choose whether to select any segments based on defocus. (default: False)
  --astigmatism_select_option, --ast
                        Choose whether to select any segments based on astigmatism. (default: False)
  --projection_correlation_select_option, --pro
                        Choose whether to select any segments based on matched cross-correlation coefficient. (default: False)
  --out-of-plane_tilt_select_option, --out
                        Choose whether to select any segments based on out-of-plane tilt angle. (default: False)
  --shift_normal_to_helix_select_option, --shi
                        Choose whether to select any segments based on forward shift difference normal to helix. This parameter tends to correlate
                        with resolution. (default: False)
  --keep_intermediate_files, --kee
                        Keep intermediate projection and diagnostic images (power spectra and reprojections) which are iteratively generated - EM
                        image stacks are deleted otherwise. (default: False)
  --symmetrize_helix, --sym
                        Tick to impose helical symmetry. Symmetry-related views are incorporated into final reconstruction. (default: False)
  --force_helical_continuity, --for
                        Tick to enforce helical continuity of segments. This way segments are removed when polarity flips within helix. Running
                        averages of in-plane rotation, shifts perpendicular to helix axis and out-of-plane angles are computed and monitored. Forward
                        differences and polarity distribution are given in statistical evaluation. (default: False)
  --limit_in-plane_rotation, --lim
                        Restricts in-plane rotation alignment. (default: False)
  --choose_out-of-plane_tilt_amplitude_correlation, --cho
                        Tick to manually to choose out-of-plane tilt range for computing sum of power spectra. This operation is relevant if you have
                        very few segments or an unexpected out-of-plane tilt distribution. Otherwise no need to change it. (default: False)
  --3d_ctf_correction, --3d
                        Tick to perform 3D CTF correction by dividing through the average of all CTFs squared. Only applied in case segments were
                        convolved by CTF. Therefore, if segments were phase-flipped no 3D CTF correction will be performed. (default: False)
  --mpi_option, --mpi   OpenMPI installed (mpirun). (default: False)

Program flow¶

project_through_reference_volume_in_helical_perspectives: Projection through reference volume
window_and_mask_input_stack: Window and mask image stack for alignment
unbend_window_and_mask_input_stack: Unbend and window image stack for alignment and reconstruction
perform_coarse_and_fine_projection_matching: Projection matching of references against image stack
select_segments_based_on_specified_criteria: Select segments according to specified criteria
apply_orientation_parameters_and_reconstruct_imposing_helical_symmetry: Reconstruct 3D volume from images using orientation parameters and helical symmetry
evaluate_alignment_parameters_and_summarize_in_plot: Evaluate alignment parameters and summarize in diagnostic plot

Functions¶

Documented functions with links to source


Input: Image input stack refinement	Output: Output volume name