Micrograph Membrane Subtraction¶

1 Basic Idea¶

After the previous step of removing membrane signals from all particles, you can put these membrane-subtracted particles back into the original micrographs to obtain new micrographs with the membrane signals removed. These micrographs can be used for subsequent processing, like re-picking the membrane proteins in them.

For each particle \(F_{RI}(x,y)\), we have a particle with membrane signals removed, \(F_{SRI}(x,y)\);
Based on the positional information of particles provided by cryoSPARC, we can replace the original particle \(F_{MI}(x,y)\) in the micrograph with \(F_{SRI}(x,y)\) to obtain a micrograph with membrane signals removed;
Of course, during the replacement process, considerations should be given to ensuring that \(F_{SRI}(x,y)\) is at the same scale as the original \(F_{RI}(x,y)\) and to how to deal with overlapping areas.

2 Specific Steps¶

2.1 Open the Interface¶

First, open the MemXTerminator main program, select the Bezierfit mode, then choose Micrograph Membrane Subtraction, and enter the Micrograph Membrane Subtraction interface:

Micrograph Membrane Subtraction interface

2.2 Parameter Explanation¶

The Micrograph Membrane Subtraction interface is as follows:

Micrograph Membrane Subtraction main interface

In the interface, you need to enter the following file path:

Particles selected starfile: Choose the STAR file that saves all particle + micrograph information.
- Usually this is particles_selected.star.
- If Micrograph Membrane Subtraction is run while particle subtraction is still running, micrographs whose dependency stacks are not available yet will be skipped and can be processed by re-running later.

In the interface, you can set the following parameters:

Cpus: You can set multiple CPUs for computation. Default is 15. However, if your GPU memory is limited, it is advised not to set too many CPUs to avoid memory issues;
Batch size: You can set how many micrographs are processed in parallel. Default is 30, twice the number of CPUs. It is recommended to set it as a multiple of the CPU number. If your GPU memory is limited, it is advised not to set a large Batch size.

After setting the appropriate parameters, click Launch to begin the membrane signal removal from the micrograph.

How to resume from a breakpoint

MemXTerminator resumes using per-output .mxt sidecar files written next to each output micrograph in the subtracted/ folder (for example, xxx_subtracted.mrc + xxx_subtracted.mrc.mxt).

mms_run_data.log is a human-readable log only and can be deleted without affecting resume.
If you already have *_subtracted.mrc outputs from an older run but no .mxt files, advanced CLI users can use --adopt_existing_outputs to backfill .mxt sidecars without recomputing.

3 Results¶

You will find a subtracted folder next to your folder containing the original micrographs, like this:

Sxxx/
├── motioncorrected/
├── subtracted/

In the subtracted folder, you will find all the .mrc files of the micrographs with membrane signals removed. You can proceed with further processing using cryoSPARC.

Note

The orginal micrographs don't have to be in the motioncorrected folder. The software will take the second folder in the directory as the original micrographs folder. For exmaple, in the particles_selected.star file, the rlnMicrographName column contains the path like Jxxx/import/xxxmicrograph.mrc. Then the software will take the import folder as the original micrographs folder and create a subtracted folder next to it.

Running from a Different Directory (CLI)

STAR files often contain relative paths like J220/import/.... If you run the CLI from a directory other than your CryoSPARC project root, use the --input_base_dir flag to specify where these relative paths should be resolved from:

python -m memxterminator.bezierfit.bin.micrograph_mem_subtract_main ... --input_base_dir /path/to/cryosparc_project

The batch scheduler handles this automatically (see Batch Scheduler).