3D Clustering - SORT3D: Sort 3D heterogeneity based on the reproducible members of K-means and Equal K-means classification. It runs after 3D refinement where the alignment parameters are determined.

Usage in command line

sxsort3d.py  stack  outdir  mask  --focus=3Dmask  --ir=inner_radius  --radius=outer_radius  --maxit=max_iter  --rs=ring_step  --xr=xr  --yr=yr  --ts=ts  --delta=angular_step  --an=angular_neighborhood  --center=centring_method  --nassign=nassign  --nrefine=nrefine  --CTF  --stoprnct=stop_percent  --sym=c1  --function=user_function  --independent=indenpendent_runs  --number_of_images_per_group=number_of_images_per_group  --low_pass_filter=low_pass_filter  --nxinit=nxinit  --unaccounted  --seed=random_seed  --smallest_group=smallest_group  --sausage  --chunk0=CHUNK0_FILE_NAME  --chunk1=CHUNK1_FILE_NAME  --PWadjustment=PWadjustment  --protein_shape=protein_shape  --upscale=upscale  --wn=wn --interpolation=method

sxsort3d exists only in MPI version.

mpirun -np 176 --host n1,n5,n6,n8,n9,n10,n0,n4,n3,n7 sxsort3d.py bdb:data sort3d_outdir1 mask.hdf --focus=ribosome_focus.hdf --chunkdir=/data/n10/pawel/ribosome_frank/ri3/main013 --radius=52 --CTF --number_of_images_per_group=2000 --low_pass_filter=.125 --stoprnct=5 

stack

Input images stack: (default required string)

mask

3D mask: (default none)

focus

Focus 3D mask: Mask used for focused clustering (default none)

radius

Outer radius for rotational correlation [Pixels]: Must be smaller than half the box size. Please set to the radius of the particle. (default -1)

delta

Angular step for projections: (default '2')

CTF

Use CTF: Do a full CTF correction during the alignment. (default False)

sym

Point-group symmetry: (default c1)

number_of_images_per_group

Images per group: Critical number of images per group, defined by user. (default 1000)

nxinit

Initial image size for sorting: (default 64)

smallest_group

Smallest group size: Minimum members for identified group. (default 500)

chunk0

Chunk file name for 1st subset: Name of chunk file containing particle IDs of 1st subset (chunk0) for computing margin of error. (default none)

chunk1

Chunk file name for 2nd subset: Name of chunk file containing particle IDs of 2nd subset (chunk0) for computing margin of error. (default none)

ir

Inner radius for rotational correlation [Pixels]: Must be bigger than 1. (default 1)

maxit

Maximum iterations: (default 25)

rs

Step between rings in rotational correlation: Must be bigger than 0. (default 1)

xr

X search range [Pixels]: The translational search range in the x direction will take place in a +/xr range. (default 1)

yr

Y search range [Pixels]: The translational search range in the y direction. If omitted it will be set as xr. (default -1)

ts

Translational search step [Pixels]: The search will be performed in -xr, -xr+ts, 0, xr-ts, xr, can be fractional. (default 0.25)

an

Local angular search width [Degrees]: This defines the neighbourhood where the local angular search will be performed. (default '-1')

center

Centering method: 0 - if you do not want the volume to be centered, 1 - center the volume using the center of gravity. (default 0)

nassign

Number of reassignment iterations: Performed for each angular step. (default 1)

nrefine

Number of alignment iterations: Performed for each angular step. (default 0)

stoprnct

Assignment convergence threshold [%]: Used to asses convergence of the run. It is the minimum percentage of assignment change required to stop the run. (default 3.0)

function

Reference preparation function: Function used to prepare the reference volume. (default do_volume_mrk02)

independent

Number of independent runs: Number of independent equal-Kmeans(default 3)

low_pass_filter

Low-pass filter frequency [1/Pixel]: Low-pass filter used for the 3D sorting on the original image size. (default -1.0)

unaccounted

Reconstruct unaccounted images: (default False)

seed

Seed: Seed used for the initial random assignment for EQ Kmeans. The program generates a random integer by default. (default -1)

sausage

Use sausage filter: (default False)

PWadjustment

Power spectrum reference: Text file containing a 1D reference power spectrum. (default none)

protein_shape

Protein Shape: It defines protein preferred orientation angles. “g” is for globular proteins and “f” is for filament proteins. (default 'g')

upscale

Power spectrum adjustment strength: This parameters adjusts how strongly the power spectrum of the volume should be modified to match the reference. A value of 1 brings the volume's power spectrum completely to the reference, while a value of 0 means no modification. (default 0.5)

wn

Target image size [Pixels]: If different than 0, then the images will be rescaled to fit this size. (default 0)

interpolation

3D interpolation method: Method interpolation in 3D. Options are tr1 or 4nn. (default '4nn')

outdir

Output directory: There is a log.txt that describes the sequences of computations in the program. (default required string)

The clustering algorithm in the program combines a couple of computational techniques, equal-Kmeans clustering, K-means clustering, and reproducibility of clustering such that it not only has a strong ability but also a high efficiency to sort out heterogeneity of cryo-EM images. The command sxsort3d.py is the protocol I {P1). In this protocol, the user defines the group size and thus defines the number of group K. Then the total data is randomly assigned into K group and an equal-size K-means (size restricted K-means) is carried out. N independent equal-Kmeans runs would give N partition of the K groups assignment. Then two-way comparison of these partitions gives the reproducible number of particles.

Zhong Huang

Category 1:: APPLICATIONS

applications.py

sxrsort3d

stable while under development:: Two programs (P1,P2) have been tested on both simulated and experimental ribosome data. For experimental ribosome data, P2 has a reproducible ratio-70-90%. P2 can 100%separate two conformations from the simulated ribosome data that contains 5 conformations.

None.