subtom_alignment

The main pipeline process script of subTOM. Iteratively aligns and averages a collection of subvolumes.

This subtomogram alignment script uses five MATLAB compiled scripts below:

• subtom_scan_angles_exact

• subtom_cat_motls

• subtom_parallel_sums

• subtom_weighted_average

• subtom_compare_motls

Options

Directories

scratch_dir

Absolute path to the folder with the input to be processed. Other paths are relative to this one.

local_dir

Absolute path to the folder on a group share, if the scratch directory is cleaned and deleted regularly this can set a local directory to which the important results will be copied. If this is not needed it can be skipped with the option skip_local_copy below.

mcr_cache_dir

Absolute path to MCR directory for the processing.

exec_dir

Directory for executables

Variables

align_exec

Alignment executable

cat_exec

Concatenate MOTLs executable

sum_exec

Parallel Summing executable

avg_exec

Weighted Averaging executable

compare_exec

Compare MOTLs executable

Memory Options

mem_free_ali

The amount of memory the job requires for alignment. This variable determines whether a number of CPUs will be requested to be dedicated for each job. At 24G, one half of the CPUs on a node will be dedicated for each of the processes (12 CPUs). At 48G, all of the CPUs on the node will be dedicated for each of the processes (24 CPUs).

mem_max_ali

The upper bound on the amount of memory the alignment job is allowed to use. If any of the processes request or require more memory than this, the queue will kill the process. This is more of an option for safety of the cluster to prevent the user from crashing the cluster requesting too much memory.

mem_free_avg

The amount of memory the job requires for averaging.

mem_max_avg

The upper bound on the amount of memory the averaging job is allowed to use.

Other Cluster Options

job_name

The job name prefix that will be used for the cluster submission scripts, log files, and error logs for the processing. Be careful that this name is unique because previous submission scripts, logs, and error logs with the same job name prefix will be overwritten in the case of a name collision.

array_max

The maximum number of jobs per cluster submission script. Cluster submission scripts work using the array feature common to queuing systems, and this value is the maximum array size used in a script. If the user requests more batches of processing than this value, then the submission scripts will be split into files of up to array_max jobs.

max_jobs

The maximum number of jobs for alignment. If the number of batches / exceeds this value the script will immediately quit.

run_local

If the user wants to skip the cluster and run the job locally, this value should be set to 1.

skip_local_copy

Set this option to 1 to skip the copying of data to local_dir.

Subtomogram Alignment Workflow Options

Parallelization Options

start_iteration

The index of the reference to start from : input will be ref_fn_prefix_start_iteration.em and all_motl_fn_prefix_start_iteration.em (define as integer e.g. start_iteration=3)

More on iterations since they’re confusing and it is slightly different here than from previous iterations.

The start_iteration is the beginning for the iteration variable used throughout this script. Iteration refers to iteration that is used for subtomogram alignment. So if start_iteration is 1, then subtomogram alignment will work using allmotl_1.em and ref_1.em. The output from alignment will be particle motls for the next iteration. This in the script is avg_iteration variable. The particle motls will be joined to form allmotl_2.em and then the parallel averaging will form ref_2.em and then the loop is done and iteration will become 2 and avg_iteration will become 3.

iterations

Number iterations (big loop) to run: final output will be ref_fn_prefix_start_iteration+iterations.em and all_motl_fn_prefix_start_iteration+iterations.em

num_ali_batch

The number of batches to split the parallel subtomogram alignment job into.

num_avg_batch

The number of batches to split the parallel subtomogram averaging job into.

File Options

all_motl_fn_prefix

Relative path and name of the concatenated motivelist of all particles (e.g. allmotl_iter.em , the variable will be written as a string e.g. all_motl_fn_prefix=’sub-directory/allmotl’)

ref_fn_prefix

Relative path and name of the reference volumes (e.g. ref_iter.em , the variable will be written as a string e.g. ref_fn_prefix=’sub-directory/ref’)

ptcl_fn_prefix

Relative path and name of the subtomograms (e.g. part_n.em , the variable will be written as a string e.g. ptcl_fn_prefix=’sub-directory/part’)

align_mask_fn

Relative path and name of the alignment mask Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

cc_mask_fn

Relative path and name of the cross-correlation mask this defines the maximum shifts in each direction Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

weight_fn_prefix

Relative path and name of the weight file.

weight_sum_fn_prefix

Relative path and name of the partial weight files.

Alignment and Averaging Options

tomo_row

Which row in the motl file contains the correct tomogram number. Usually row 5 and 7 both correspond to the correct value and can be used interchangeably, but there are instances when 5 contains a sequential ordered value starting from 1, while 7 contains the correct corresponding tomogram.

apply_weight

Apply weight to subtomograms (1=yes, 0=no).

apply_mask

Apply mask to subtomograms (1=yes, 0=no).

psi_angle_step

Angular increment in degrees, applied during the cone-search, i.e. psi and theta (define as real e.g. psi_angle_step=3). Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

psi_angle_shells

Number of angular iterations, applied to psi and theta (define as integer e.g. psi_angle_shells=4). Note that in terms of cones this is twice the number of cones sampled. Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

phi_angle_step

Angular increment for phi in degrees, (define as real e.g. phi_angle_step=3). Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

phi_angle_shells

Number of angular iterations for phi, (define as integer e.g. phi_angle_shells=6). Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

high_pass_fp

High pass filter cutoff (in transform units (pixels): calculate as (box_size * pixelsize) / (resolution_real) (define as integer). Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

high_pass_sigma

High pass filter falloff sigma (in transform units (pixels): describes a Gaussian sigma for the falloff of the high-pass filter past the cutoff above. Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

low_pass_fp

Low pass filter (in transform units (pixels): calculate as (box_size * pixelsize) / (resolution_real) (define as integer). Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

low_pass_sigma

Low pass filter falloff sigma (in transform units (pixels): describes a Gaussian sigma for the falloff of the low-pass filter past the cutoff above. Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

nfold

Symmetry, if no symmetry nfold=1 (define as integer e.g. nfold=3). Leave the parentheses and if the number of values is less than the number of iterations the last value will be repeated to the correct length.

threshold

Threshold for cross correlation coefficient. Only particles with ccc_new > threshold will be added to new average (define as real e.g. threshold=0.5). These particles will still be aligned at each iteration.

iclass

Particles with that number in position 20 of motivelist will be added to new average (define as integer e.g. iclass=1). NOTES: Class 1 is ALWAYS added. Negative classes and class 2 are never added.

Example

scratch_dir="${PWD}"

local_dir=""

mcr_cache_dir="${scratch_dir}/mcr"

exec_dir="/net/dstore2/teraraid/dmorado/software/subTOM/bin"

align_exec="${exec_dir}/alignment/subtom_scan_angles_exact"

cat_exec="${exec_dir}/MOTL/subtom_cat_motls"

sum_exec="${exec_dir}/alignment/subtom_parallel_sums"

avg_exec="${exec_dir}/alignment/subtom_weighted_average"

compare_exec="${exec_dir}/MOTL/subtom_compare_motls"

mem_free_ali=1G

mem_max_ali=64G

mem_free_avg=1G

mem_max_avg=64G

job_name=subTOM

array_max=1000

max_jobs=4000

run_local=0

skip_local_copy=1

start_iteration=1

iterations=3

num_ali_batch=1

num_avg_batch=1

all_motl_fn_prefix="combinedmotl/allmotl"

ref_fn_prefix="ref/ref"

ptcl_fn_prefix="subtomograms/subtomo"

align_mask_fn=("otherinputs/align_mask_1.em" \
               "otherinputs/align_mask_2.em" \
               "otherinputs/align_mask_3.em")

cc_mask_fn=("otherinputs/cc_mask_r10.em" \
            "otherinputs/cc_mask_r05.em")

weight_fn_prefix="otherinputs/ampspec"

weight_sum_fn_prefix="otherinputs/wei"

tomo_row=7

apply_weight=0

apply_mask=1

psi_angle_step=(10 5 2.5)

psi_angle_shells=(4)

phi_angle_step=(20 5)

phi_angle_shells=(6)

high_pass_fp=(1)

high_pass_sigma=(2)

low_pass_fp=(12 15 18)

low_pass_sigma=(3)

nfold=(1 6)

threshold=-1

iclass=0