Thank you for the suggestions, Oli!
- I tried the CPU-based extraction job as you suggested, and it give the same results
2.The auto-calculated mask during reconstruction is poor, presumably because it’s calculated off the reconstructed volume. Based off your suggestion, I tried supplying the mask that I used for the Local Refinement before re-extracting. Although the reported resolution improved from 19-25 A to 15 A, the quality of the actual reconstruction hasn’t changed. As before, at high thresholds, the map looks like an aggressively low-pass filtered version of the original map, but at low thresholds, the map looks weirdly elongated along one axis. I’ve verified that the auto-generated mask and the mask that I’ve supplied are both positioned correctly by opening them in ChimeraX - I tried extracting without Fourier cropping, and still have the same issue.
Yes, I’m extracting the from the same set of micrographs in cryoSPARC.
So based off the troubleshooting I’ve done so far, it seems like I’ve narrowed the problem down to the actual extraction job. Everything looks fine before re-extracting, and reconstructing the map directly after the initial Local Refinement or directly after the Volume Align Tools results in a good reconstruction. However, after any type of Extraction job (GPU or CPU, Fourier cropping or not, larger box size or same box size), the alignments degrade. Based off the bimodal distribution in the alignment changes plot in my original post, the extraction job appears to preserve the alignments for half the particles, but completely scrambles the alignments for the other half…
For now, I can sidestep this issue by doing a global refinement after re-extracting followed by a Local Refinement. This allows me to achieve the same resolution that I attained prior to re-extracting, but I’m really confused why the extraction job is causing issues for this dataset and not any of the others I’ve processed. If I have time, I might try to inspect the .cs file for the Local Refinement and see if there’s anything weird with the population of particles with scrambled alignments. Maybe they all come from some corrupted batch of micrographs? I’d really appreciate any other suggestions for what I could look for.
Cheers,
cbeck