automated masking during refinement e2spt_refine_new.py

[Adam Schröfel]

Hi all, I have a question regarding the the automatic mask generation during the new refinement step. I have geberated initial model from the particles - it looks fine and contains the whole microtubule doublet. However, after refinenment, only the central tubule is masked and the second tubule (it is not complete, more flexible and therefore faint) is not averaged. Is there any possibility to tune the automasking (e.g. some thresholds...?) or I should make and provide a binary mask instead?

Thanks for the suggestions and help

Adam

[Ludtke, Steven J.]

Hi Adam,

if you run 

e2spt_refine_new.py --help

(or look at the Help tab in the GUI, though formatting isn't always great), you'll see there are several options

related to masking:

  --mask MASK           Mask applied to the results (instead of automasking)

  --automaskexpand AUTOMASKEXPAND

                        Default=boxsize/20. Specify number of voxels to expand mask before soft edge.

  --maskalign MASKALIGN

                        Mask file applied to 3D alignment reference in each iteration. Not applied to the average, which will follow

                        normal masking routine.

The automasking works fairly well for discrete objects like ribosomes, but can have thresholding issues in some cases making part of the structure disconnected and getting cut off like this. While --automaskexpand is a simple solution which will help in cases where low occupancy ligands get cut off, in a case like yours, I would suggest making your own mask with e2filtertool.py and using that. Just make sure it's a proper 'soft' edged mask.

It's a bit dated, but this tutorial shows how to use e2filtertool in general. To make a mask, you would normally use a sequence of filter.lowpass.gauss, threshold.binary to construct a mask. ie - you blur the map a bit to low resolution, pick a threshold which includes all of the density you're interested in and threshold. Low pass filter that and threshold again to expand the mask a bit away from the surface, threshold again, then apply a final low pass filter to give it a soft edge.

---

Steven Ludtke, Ph.D. <slu...@bcm.edu>                      Baylor College of Medicine
Charles C. Bell Jr., Professor of Structural Biology        Dept. of Biochemistry 
Deputy Director, Advanced Technology Cores                  and Molecular Pharmacology
Academic Director, CryoEM Core
Co-Director CIBR Center

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<initial_model.JPG><refinement.JPG>

[Adam Schröfel]

Thanks Steve, 

I did the mask and it seems to be working!

Best regards

Adam

Dne pondělí 4. září 2023 v 15:32:06 UTC+2 uživatel Ludtke, Steven J. napsal: