I removed signal from the detergent belt surrounding a membrane protein.
After local refinement, I can still see a faint signal where the detergent belt used to be. Interestingly for me, the location of this boundary is the same as the one for non-subtracted particles.
On the cryosparc web page (https://guide.cryosparc.com/processing-data/all-job-types-in-cryosparc/local-refinement/job-particle-subtraction-beta), it is written in the “limitations”:
For molecules with significant flexibility, Particle Subtraction is only an approximation of the true signal subtracted images, due to the fact that particle poses are assumed constant and are not iteratively refined as in Local Refinement.
The one thing that detergent belts possess is flexibility, and are thus not constant at the boundary typically seen after reconstruction/refinement.
Could it be the reason for the “persistence” of detergent belt signal at this specific boundary ?
Is there a reference for this in the literature ? A reference describing what is said in the “limitations”?
Thank you for your help.
The detergent belt in each original particles image is heterogeneous. It does not contain a well defined signal comparing to the protein molecule. Subtract detergent signal from the refined structure therefore will not completely eliminate detergent signal in each original images. In my opinion.
Yes, this is my hunch, but I’m looking for proofs if it has been done out there?
A useful reference that has a brief section on partial signal subtraction is Prof. Scheres’s chapter in Methods in Enzymology, Processing of Structurally Heterogeneous Cryo-EM Data in RELION. It mentions the limitation in that signal subtraction makes the assumption that the current poses are correct, thus any inaccuracies in the poses will result in subtracting slightly mis-aligned references. The more accurate the alignments are, the better the subtraction will work, so it can be beneficial to do subtraction after a round of local refinement (Oli described this workflow nicely in this thread).
Independent of that issue, @zhenyu_tan’s comment on heterogeneity is the most likely explanation behind why you are seeing residual density. Each image contains a different micelle due to disorder, which breaks the assumption behind subtraction (i.e. that each image contains a discrete number of rigid bodies). For that reason, we typically don’t recommend using subtraction for micelles (see here for another thread on this topic).