Segmentalign2d¶
Program to align segments from helical specimens with a restrained in-plane rotation of 0 or 180 +/- delta degrees
Input: Image input stack
Output: Image output stack
Parameters¶
Parameter |
Example (default) |
Description |
|---|---|---|
Image input stack |
protein_stack.hdf |
Input stack: accepted file formats (hdf). |
Image output stack |
protein_stack.hdf |
Output stack: accepted file formats (hdf). |
Reference option |
True |
Provide a stack of images as a reference. If not ticked, a simple average of the provided image stack will be used. |
Image reference stack |
protein_reference_stack.hdf |
Input reference stack: accepted file formats (spi, .hdf, .img, .hed). |
Update references |
True |
Check if references should be updated for every iteration. This is useful if you have a high ratio of images / references and you expect to improve your references. In case you simply want to match or assign having a low images / references ratio you should turn it off. |
Aligned average stack |
protein_reference_aligned.hdf |
Output aligned reference stack: accepted file formats (spi, .hdf, .img, .hed). |
Pixel size in Angstrom |
1.163 |
Pixel size is an imaging parameter (accepted values min=0.001, max=100). |
Estimated helix width and height in Angstrom |
(200, 600) |
Generous width and height measure of helix required for rectangular mask (accepted values min=20, max=1500). |
Sample parameter file¶
You may run the program in the command line by providing the parameters via a text file:
segmentalign2d --f parameterfile.txt
Where the format of the parameters is:
Image input stack = protein_stack.hdf
Image output stack = protein_stack.hdf
Reference option = True
Image reference stack = protein_reference_stack.hdf
Update references = True
Aligned average stack = protein_reference_aligned.hdf
Pixel size in Angstrom = 1.163
Estimated helix width and height in Angstrom = (200, 600)
Additional parameters (intermediate level)¶
Parameter |
Example (default) |
Description |
|---|---|---|
Number of iterations |
2 |
Number of iteration cycles of multi-reference alignment. Internally imageswill be binned to speed up processing (accepted values min=1, max=200). |
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). |
Internal binning factor |
2 |
In order to speed up alignments images can be optionally binned (accepted values min=1, max=20). |
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). |
X and Y translation range in Angstrom |
(50, 50) |
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). |
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). |
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:
segmentalign2d --f parameterfile.txt
Where the format of the parameters is:
Image input stack = protein_stack.hdf
Image output stack = protein_stack.hdf
Number of iterations = 2
Reference option = True
Image reference stack = protein_reference_stack.hdf
Update references = True
Aligned average stack = protein_reference_aligned.hdf
Pixel size in Angstrom = 1.163
Estimated helix width and height in Angstrom = (200, 600)
Absolute X and Y translation limit in Angstrom = (100, 100)
Internal binning factor = 2
Limit in-plane rotation = True
Delta in-plane rotation angle = 10.0
X and Y translation range in Angstrom = (50, 50)
High-pass filter option = False
Low-pass filter option = True
High and low-pass filter cutoffs in 1/Angstrom = (0.001, 0.09)
MPI option = True
Number of CPUs = 8
Temporary directory = /tmp
Additional parameters (expert level)¶
Parameter |
Example (default) |
Description |
|---|---|---|
Local refinement |
False |
This option can be used to refine locally around the alignment parameters stored in the header of the image stack. |
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 filter option |
True |
Automatic filter design derived from decay of Fourier ring correlation between images. |
Sample parameter file (expert level)¶
You may run the program in the command line by providing the parameters via a text file:
segmentalign2d --f parameterfile.txt
Where the format of the parameters is:
Image input stack = protein_stack.hdf
Image output stack = protein_stack.hdf
Number of iterations = 2
Reference option = True
Image reference stack = protein_reference_stack.hdf
Update references = True
Aligned average stack = protein_reference_aligned.hdf
Pixel size in Angstrom = 1.163
Estimated helix width and height in Angstrom = (200, 600)
Local refinement = False
Absolute X and Y translation limit in Angstrom = (100, 100)
Internal binning factor = 2
Limit in-plane rotation = True
Delta in-plane rotation angle = 10.0
X and Y translation range in Angstrom = (50, 50)
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 filter option = True
MPI option = True
Number of CPUs = 8
Temporary directory = /tmp
Command line options¶
When invoking segmentalign2d, you may specify any of these options:
usage: segmentalign2d [-h] [--g] [--p] [--f FILENAME] [--c] [--l LOGFILENAME] [--d DIRECTORY_NAME] [--version] [--reference_option]
[--update_references] [--local_refinement] [--limit_in-plane_rotation] [--high-pass_filter_option] [--low-pass_filter_option]
[--custom_filter_option] [--automatic_filter_option] [--mpi_option]
[input_output [input_output ...]]
Program to align segments from helical specimens with a restrained in-plane rotation of 0 or 180 +/- delta degrees
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_option, --ref
Provide a stack of images as a reference. If not ticked, a simple average of the provided image stack will be used. (default:
False)
--update_references, --upd
Check if references should be updated for every iteration. This is useful if you have a high ratio of images / references and
you expect to improve your references. In case you simply want to match or assign having a low images / references ratio you
should turn it off. (default: False)
--local_refinement, --loc
This option can be used to refine locally around the alignment parameters stored in the header of the image stack. (default:
False)
--limit_in-plane_rotation, --lim
Restricts in-plane rotation alignment. (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_filter_option, --aut
Automatic filter design derived from decay of Fourier ring correlation between images. (default: False)
--mpi_option, --mpi OpenMPI installed (mpirun). (default: False)
Program flow¶
prepare_alignment: Prepare alignment
prepare_reference_images_for_alignment: Prepare reference images for alignment
align_images_to_references: Aligns images to reference
pass_alignment_parameters_from_reference_groups_to_images: Apply alignment parameters to images