I am working on a complex of the 40S ribosomal subunit with different semi-flexible components attached. I have had good results disentangling flexibility with 3DVA clustering, but then when I try to refine the clusters furthers, the 40S dominates the alignments and my smaller protein gets blurry again.
Running local refinement helped a little bit, but I want to perform particle subtraction on most of the 40S to further improve the input particles for the local refinement.
I prepared a mask (using segger in chimera - which worked well to generate masks for the 3DVA), resampled on a correct volume, imported it, dilated by 5 and padded by 5 using the volume tools, but then the particle subtraction runs and the particles look the same as before.
I went through the forums and found the suggestion to run a local refinement with the subtraction mask before subtraction, which I did, but nothing changed.
I tried running a 2D classification and a 3D homogeneous refinement with the supposedly subtracted particles, and in both cases the results look the same as before the particle subtraction.
I went through the parameters of the particle subtraction job, but there is not really that much to change (to my inexperienced eyes).
We are running cryosparc 3.1.