The code in this project is distributed under the GPL-3.0 license.
Post-processing of cryo-EM maps and generation of FSC curves corrected by masking effects
NOTE: The code in postprocessing.py was adapted from https://github.com/C-CINA/focustools.
At a minimum, two half maps should be provided as input. To see all available options type:
./postprocessing.py --help
Usage: postprocessing.py [] [options]
Options:
-h, --help show this help message and exit
--out=postprocess Output rootname.
--angpix=1.0 Pixel size in Angstroems
--fsc_threshold=0.143
Display the resolution at which the FSC curve crosses
this value.
--lowpass="auto" Resolution (in Angstroems) at which to low-pass filter
the final map. A negative value will skip low-pass
filtering. Default ('auto') is to low-pass at
resolution determined from FSC threshold.
--highpass="auto" Resolution (in Angstroems) at which to high-pass
filter the final map. Useful to to attenuate artefacts
such as background ramps (e.g. high-pass at 2/3 of the
box size). A negative value will skip high-pass
filtering (default).
--mask=MyMask.mrc A file containing the mask to be applied to the half-
maps before calculating the FSC. Can be combined with
other masking options.
--mask_radius=0.5 Creates a soft spherical mask. This is the radius of
such mask, in pixels or fraction of the box size. Can
--automask_floodfill_center=0,0,0
Three numbers describing where the center of the
sphere should be placed to initialize flood-filling
approach in auto-masking (in pixels). Useful to mask
parts of the 3D map that are not close to the center.
Default is the middle of the box: 0,0,0. Can be
positive or negative.
--automask_floodfill_fraction=0.1
Use this fraction of the voxels with the highest
densities (0.10 = top 10 percent highest densities) to
initialize the flood-filling algorithm. Typically
follows the correct map density for capsid-like or
hollow-center particles. Note that this is only used
to initialize the smart masking algorithm and has no
influence on the threshold, that is defined
independently (see options below).
--automask_lp=14.0 Resolution (in Angstroems) at which to low-pass filter
the input map for auto-masking purposes. Should
typically be in the range of 10-20 A. However if using
the flood-filling approach a value in the range 5-10 A
might work better. Use a negative number to disable
this filter.
--automask_lp_edge_width=5.0
Width of the cosine-edge low-pass filter to be used
for auto-masking (in Fourier pixels).
--automask_lp_gauss Use a Gaussian instead of cosine-edge low-pass filter
for auto-masking.
--automask_threshold=0.02
Absolute threshold to generate the initial binary
volume in auto-masking. This has precedence over
--automask_fraction and --automask_sigma.
--automask_fraction=0.1
Use this fraction of the voxels with the highest
densities (0.10 = top 10 percent highest densities) to
generate the initial binary volume in auto-masking.
This has precedence over --automask_sigma.
--automask_sigma=1.0 Use this many standard deviations above the mean
density value as threshold for initial binary volume
generation in auto-masking. This is the default
option.
--automask_expand_width=3.0
Width in pixels to expand the binary mask in auto-
masking. Useful to make the mask more generous and
correct for imperfections of the initial binarization.
--automask_soft_width=6.0
Width in pixels to expand the binary mask in auto-
masking with a soft cosine edge. Very important to
prevent artificial correlations induced by the mask.
--automask_optimize Will generate a mask automatically based on the
automask procedure, and then tune it to optimize the
masked FSC corrected by High-Resolution Noise
Substitution (Chen et al, Ultramicroscopy 2013)
--skip_fsc Skip all FSC calculations, only do masking and
filtering stuff.
--mw=1000.0 Molecular mass in kDa of particle or helical segment
comprised within the mask. Needed to calculate volume-
normalized Single-Particle Wiener filter (Sindelar &
Grigorieff, JSB 2012). If not specified, will do
conventional FSC weighting on the final map (Rosenthal
& Henderson, JMB 2003).
--mw_ignore=MW_IGNORE
CAUTION! EXPERIMENTAL OPTION: Molecular mass in kDa
present within the mask that needs to be ignored.
Needed to calculate an adaptation of the volume-
normalized Single-Particle Wiener filter (Sindelar &
Grigorieff, JSB 2012). Only used if --mw is also
specified. May be useful if your particles are
extracted from 2D crystals.
--skip_fsc_weighting Do NOT apply FSC weighting (Rosenthal & Henderson, JMB
2003) to the final map, nor the Single-Particle Wiener
filter (Sindelar & Grigorieff, JSB 2012).
--gaussian Apply a Gaussian (instead of cosine-edge) low-pass
filter to the map, with cutoffs defined by --highpass
and --lowpass.
--cosine Apply a cosine-edge low-pass filter to the final map,
with cutoffs defined by --highpass and --lowpass. This
is the default. The width of the cosine edge can be
specified with the option --edge_width.
--cosine_edge_width=3.0
Width of the cosine-edge filter (in Fourier pixels).
The cutoff frequency will have a weight of 0.5, with
corresponding weighting above and below following the
cosine falloff. If set to zero, becomes a top-hat
filter with weighting of 1.0 at the cutoff frequency.
--tophat Apply a top-hat low-pass filter to the final map.
Equivalent to specifying --cosine with
--cosine_edge_width=0.
--mtf=MTF File containing the detector MTF for sharpening of the
final map.
--auto_bfac=10.0,0.0 Estimate B-factor automatically using information in
this resolution range, in Angstroems (lowres,highres).
This works based on the Guinier plot, which should
ideally be a straight line from ~10.0 A and beyond
(Rosenthal & Henderson, JMB 2003).'If you set lowres
and/or maxres to -1, these values will be calculated
automatically. MTF and FSC weighting information are
employed, if not ommitted.
--adhoc_bfac=ADHOC_BFAC
Apply an ad-hoc B-factor to the map (in Angstroems^2).
Can be positive (smoothing) or negative (sharpening).
--whiten Whiten the final map (that is, makes the radial
Amplitude Spectrum equal to 1.0 across all
frequencies). Should normally be used in combination
with the option --gaussian and --skip_fsc_weighting,
and also --highpass in some cases. If using this
option, B-factor and MTF sharpening become
meaningless.
--whiten_amps Whiten the radial Amplitude Spectrum instead of the
radial Power Spectrum (Amplitudes^2). Whitening
amplitudes seems to give a slightly more peaky (sharp)
map as measured by kurtosis, but the results are
visually almost identical. Only valid if using option
--whiten above.
--randomize_below_fsc=0.8
If provided, will randomize phases for all Fourier
shells beyond the point where the FSC drops below this
value, to assess correlations introduced by the mask
by High-Resolution Noise Substitution (Chen et al,
Ultramicroscopy 2013). Be aware that this procedure
may introduce a 'dip' in the FSC curves at the
corresponding resolution value, but that is normal.
--randomize_beyond=10
If provided, will randomize phases for all Fourier
shells beyond this resolution shell, to assess
correlations introduced by the mask by High-Resolution
Noise Substitution (Chen et al, Ultramicroscopy 2013).
Be aware that this procedure may introduce a 'dip' in
the FSC curves at the corresponding resolution value,
but that is normal.
--random_seed=123456789
Choose the random seed for High-Resolution Noise
Substitution (see option --randomize_below_fsc above)
--flip_x Invert handedness of input volumes along the X axis.
--flip_y Invert handedness of input volumes along the Y axis.
--flip_z Invert handedness of input volumes along the Z axis.
--xml Save FSC curves also in XML format for EMDB
deposition.
Convert a cisTEM parameter file into a .bild file for visualization in Chimera
NOTE: The code in par_to_bild.py is based on RELION's SymList Object and pyem's star2bild script.
The program takes a .par file as input and produces a .bild file ready to load in Chimera.
par_to_bilt.py --help
usage: par_to_bild.py [-h] [--input INPUT] [--output OUTPUT] [--tomo] [--tilt_max TILT_MAX] [--healpix_order HEALPIX_ORDER] [--apix APIX] [--boxsize BOXSIZE] [--height_scale HEIGHT_SCALE] [--width_scale WIDTH_SCALE] [--occ_cutoff OCC_CUTOFF] [--sym SYM]
optional arguments:
-h, --help show this help message and exit
--input INPUT Input alignment file
--output OUTPUT Output .bild file
--tomo Refinement from tomogram tilt images
--tilt_max TILT_MAX max tilt angel to use from tomo data
--healpix_order HEALPIX_ORDER
Healpix order (Relion convention)
--apix APIX Angstroms per pixel
--boxsize BOXSIZE Box size in pixels
--height_scale HEIGHT_SCALE
--width_scale WIDTH_SCALE
--occ_cutoff OCC_CUTOFF
Only use the particles with occupancy above this threshold
--sym SYM Symmetry group to impose on distribution (Relion conventions)