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Extracting Subtomograms

In this part, we will familiarize ourselves with STOPGAP's approach to generating a parameter file using a parser, then running that file. STOPGAP jobs are run by using a task-specific parser script (named stopgap_*_parser.sh) to generate a parameter file (named *_param.star) and then running that parameter file using the run_stopgap.sh script.

The first STOPGAP task we will perform is extracting subtomograms.

Preparing Extraction Parameter File

With the lists and run script prepared, we are now ready to extract our subtomograms.

  1. Open stopgap_extract_parser.sh in a text editor.

  2. There are four blocks of options here: "Parser Options" which contains the path to the output parameter file; "Folder Options" which provides paths to folders; "File options" which are the input files for extraction; and "Extraction Parameters" which are settings for the output subtomograms.

  3. In the Parser Options, the default param_name is fine.

  4. Under Folder options, update the rootdir to the absolute path of the init_ref/ directory. The other directory parameters can be left alone; they are overrides to the standard STOPGAP structure.

  5. Under File options, update the lists.

    • motl_name='allmotl_tomo1_obj1_1.star'

    • wedgelist_name='wedgelist.star'

    • tomolist_name='sg_tomolist.txt'

    Since these are all lists, they are assumed to be in the listdir.

    NOTE: since we are providing a tomolist, tomodir is ignored and can be left as 'none'.

  6. Set the extraction parameters. The default subtomo_name is 'subtomo'. For boxsize, 32 should be sufficient here. Set pixelsize to 10.8 since we binned our 1.35 Å pixels by a factor of 8. For output_format, we find that 'mrc8' works well, this saves the subtomogram as an 8-bit .mrc file.

    NOTE: While 8-bit only provides 256 gradations, we generally find this is sufficient for the local information contained within a subtomogram. During extraction, the subtomogram is cropped and its values are floated between 0 and 255, rounded, and saved.

  7. Save and close the file. Run in the terminal.

    ./stopgap_extract_parser.sh

This will generate a new parameter file in the params/ folder. Feel free to preview it if you're curious.

Preparing to Run STOPGAP with MPI

  1. Open run_stopgap.sh in any text editor, for example, gedit using a terminal window:

    gedit run_stopgap.sh

    The main parameters here are the "run options" which manage parallelization and the "directories" block, which manages directories and paths.

  2. For parallelization parameters, set run_type to 'local', nodes to 1, n_cores to 1, and copy_local to 0. The rest of the run options are SLURM-specific and can be ignored.

  3. Set rootdir to the absolute path of your init_ref/ folder (e.g. ~/HIV_dataset/subtomo/init_ref/). We will update paramfilename before running each job.

    NOTE: Remember to end your rootdir path with a /!

  4. Set paramfilename to params/extract_param.star. Save the file and run STOPGAP by running run_stopgap.sh in a terminal.

    ./run_stopgap.sh

    NOTE: STOPGAP is setup here to run through the stopgap_watcher, which is a separate program to track STOPGAP progress. This is not required; for running on clusters where programs are not allowed to be run on submission nodes, stopgap_watcher can be run on any computer that has access to the working directory.