Particle Membrane Subtraction Using Radonfit¶

1 Basic Idea¶

After completing the previous step of Membrane Analysis Using Radonfit, you have obtained a file mem_analysis.star containing all analysis results, including membrane center coordinates, angles, membrane curvature, and so on. In this step, you will use this information to subtract membrane signals from all particles.

For each particle \(F_{RawImage}(x,y)\) (abbreviated as \(F_{RI}(x,y)\) ), corresponding to its template \(f_{2DAverage}(x,y)\), we have a membrane-averaged function \(f_{AveragedMembrane}(x,y)\) (abbreviated as \(f_{AM}(x,y)\)) and a corresponding membrane mask \(f_{MembraneMask}(x,y)\) (abbreviated as \(f_{MM}(x,y)\) );
Based on alignment information from cryoSPARC, i.e., the previously used particles_selected.star file, we can determine the displacement \((\Delta x, \Delta y)\) and rotation angle \(\psi\) for each particle. Using these parameters, transform \(f_{AM}(x,y)\) and \(f_{MM}(x,y)\) accordingly to match each particle's membrane signal, resulting in transformed functions \(f_{AM}'(x,y)\) and \(f_{MM}'(x,y)\);
For each particle, perform trajectory averaging using \(f_{AM}'(x,y)\) and \(f_{MM}'(x,y)\), thereby obtaining each particle's corresponding membrane signal \(F_{AM}(x,y)\) and mask \(F_{MM}(x,y)\);
For each particle, to obtain \(F_{SubtractedRawImage}(x,y)\) (abbreviated as \(F_{SRI}(x,y)\) ), use the following equation:

\[ F_{SRI}(x,y) = F_{RI}(x,y) - \lambda \times F_{AM}(x,y) \]

where \(\lambda\) is a constant controlling the intensity of membrane subtraction. \(\lambda\) is determined by:

\[ \lambda = \arg \min_{\lambda} || F_{RI}(x,y) \times F_{MM}(x,y) - \lambda \times F_{AM}(x,y) ||_1 \]

2 Specific Steps¶

2.1 Open the Interface¶

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

Particles Membrane Subtraction (Radonfit) interface

2.2 Set Appropriate Parameters¶

In the Particles Membrane Subtraction interface:

Particles Membrane Subtraction main interface

Particles Membrane Subtraction (Radonfit) main interface

You need to enter the following file paths:

Particles selected starfile: Choose the .star file that saves all particles information, usually particles_selected.star;
Membrane analysis results: Choose the .star file that contains all membrane analysis information, usually mem_analysis.star;

You can set the following parameters:

Bias: During membrane signal subtraction, you can set a bias value to manually adjust the strength of membrane signal removal. The actual membrane subtraction intensity \(\lambda' = \lambda + \text{bias}\). Default is 0.05;
Extra_mem_dist: When performing trajectory averaging, you can set an extra membrane distance, which adds an additional distance to the membrane to increase the stability of the subtraction effect. Default is 15;
Scaling_factor_start and Scaling_factor_end: The search range for the scaling factor (i.e., \(\lambda\)) during trajectory averaging. Default is between 0.1 and 1;
Scaling_factor_step: The search step for the scaling factor (i.e., \(\lambda\)) during trajectory averaging. Default is 0.01. A smaller step size may result in more accurate determination of the scaling factor but increases computation time;
CPU: You can set multiple CPUs for computation. Default is 10. 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 particle stacks are processed in parallel. Default is 20, 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 subtraction.

How to resume from a breakpoint

If the job is interrupted for some reason, please don't worry. You can continue to do the membrane subtraction because every time you begin the membrane subtraction, the software will read the radfit_pms_run_data.log file, which records the particle stacks that have been processed. The software will automatically skip the processed particle stacks and continue to process the remaining particle stacks.

3 Results¶

You will find a subtracted folder next to the folder where you extracted the particles, like this:

Jxxx/
├── extract/
├── subtracted/

In the subtracted folder, you will find all the mrc files of the particles with membrane signals removed. You can proceed with further processing using cryoSPARC or put the membrane-subtracted particles back to the micrographs for subsequent processing.

Warning

Please note that the software can only recognize the extract folder in the Jxxx folder, and usually in the particles_selected.star file, the rlnImageName column contains the path like Jxxx/extract/xxx.mrc. If you find that the software cannot recognize the extract folder, please check the particles_selected.star file and make sure the rlnImageName column is correct.