.. _segmultirefine3d: Segmultirefine3d ---------------- Program to iteratively refine multiple 3D structures of helical specimens competitively from segment stacks +--------------------------------------------------+--------------------------------------------------+ | |segmultirefine3d_input.png| | |segmultirefine3d_output.png| | +--------------------------------------------------+--------------------------------------------------+ | Input: Image input stack refinement | Output: Output volume name | +--------------------------------------------------+--------------------------------------------------+ .. |segmultirefine3d_input.png| image:: ../gallery_images/segmultirefine3d_input.png :alt: tree :width: 4 cm :target: ../_images/segmultirefine3d_input.png .. |segmultirefine3d_output.png| image:: ../gallery_images/segmultirefine3d_output.png :alt: tree :width: 4 cm :target: ../_images/segmultirefine3d_output.png .. _making_a_segmultirefine3d_table_for_beginner: Parameters ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .. tabularcolumns:: |p{4cm}|p{2.5cm}|p{9cm}| ============================================================================== ============================================================================== ==================================================================================================== 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). 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 symmetries in Angstrom or degrees (1.408, 22.03); (40, 60); (60, 80) List of semicolon-separated pairs of 'helical rise/pitch' (Angstrom) and 'rotation/number of units per turn' to be imposed to 3D reconstructions. 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). ============================================================================== ============================================================================== ==================================================================================================== .. _segmultirefine3d_parameter_file_for_beginner: Sample parameter file ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ You may run the program in the command line by providing the parameters via a text file:: segmultirefine3d --f parameterfile.txt Where the format of the parameters is:: Image input stack refinement = protein_stack.hdf Output volume name = recvol.hdf 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 symmetries in Angstrom or degrees = (1.408, 22.03); (40, 60); (60, 80) Image alignment size in Angstrom = 700 Step size of segmentation in Angstrom = 70 .. _making_a_segmultirefine3d_table_for_intermediate: Additional parameters (intermediate level) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .. tabularcolumns:: |p{4cm}|p{2.5cm}|p{9cm}| ============================================================================== ============================================================================== ==================================================================================================== 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 volumes reference_vol???.hdf Either single reference volume or list of references to be used for competitive 3D structure refinement. If multiple references specified header information provides helical symmetry information. 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 symmetries 1, 1, 1 List of comma-separated rotational symmetries. Position in list corresponds to order of list of 'Helical symmetries'. Helix polarities polar, apolar 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. ============================================================================== ============================================================================== ==================================================================================================== .. _segmultirefine3d_parameter_file_for_intermediate: Sample parameter file (intermediate level) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ You may run the program in the command line by providing the parameters via a text file:: segmultirefine3d --f parameterfile.txt Where the format of the parameters is:: Image input stack refinement = protein_stack.hdf Output volume name = recvol.hdf Diagnostic plot prefix = diagnostic_plot.pdf Number of iterations = 20 Reference structure option = False Reference volumes = 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 symmetries in Angstrom or degrees = (1.408, 22.03); (40, 60); (60, 80) Rotational symmetries = 1, 1, 1 Helix polarities = polar, apolar 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 .. _making_a_segmultirefine3d_table_for_expert: Additional parameters (expert level) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ .. tabularcolumns:: |p{4cm}|p{2.5cm}|p{9cm}| ============================================================================== ============================================================================== ==================================================================================================== Parameter Example (default) Description ============================================================================== ============================================================================== ==================================================================================================== 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). ============================================================================== ============================================================================== ==================================================================================================== .. _segmultirefine3d_parameter_file_for_expert: Sample parameter file (expert level) ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ You may run the program in the command line by providing the parameters via a text file:: segmultirefine3d --f parameterfile.txt Where the format of the parameters is:: Image input stack refinement = protein_stack.hdf Output volume name = recvol.hdf Diagnostic plot prefix = diagnostic_plot.pdf Number of iterations = 20 Reference structure option = False Reference volumes = 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 symmetries in Angstrom or degrees = (1.408, 22.03); (40, 60); (60, 80) Rotational symmetries = 1, 1, 1 Helix polarities = polar, apolar 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 .. _using_segmultirefine3d_on_cmdline: Command line options ~~~~~~~~~~~~~~~~~~~~~~~ When invoking segmultirefine3d, you may specify any of these options:: usage: segmultirefine3d [-h] [--g] [--p] [--f FILENAME] [--c] [--l LOGFILENAME] [--d DIRECTORY_NAME] [--version] [--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 iteratively refine multiple 3D structures of helical specimens competitively from segment stacks 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 --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 ~~~~~~~~~~~~~~~~~~~~ .. toctree:: :maxdepth: 1 segmultirefine3d_functions