In order to do signal subtraction I low-passed a refined map, used the volume eraser to remove the region I eventually wanted to focus on, and made a mask out of the remaining non-interesting region. Before applying that mask to the refined particles I used relion_image_handler to subtract the region just used to make a mask (unwanted region) from the entire low-passed map, giving me a low-passed region of interest. I make both of these maps into masks (Mask creation in RELION). Thus I have two masks: #1 - around the region of interest and #2 - around the remaining mass of the protein.
After the signal subtraction using the mask around the unwanted region (#2), I apply the mask around the region of interest (#1) to these subtracted particles in a refinement.
After the refinement I looked at the structure and something weird happened: the region of interest is well-defined with a gap around it (where the applied mask would be), but then there is a lot of noisy mass with the general shape of the rest of the protein I subtracted out. What would be the cause of this?
To verify I did signal subtraction correctly, I went through another series of jobs we did on a different subset of the same original data set and the refinement of that data after subtraction does not have that noise outside the mask region.
How do I go about overcoming this?
Austin Dixon
Rockefeller University
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