Extraction (and box size cropping in the Downsample job) were done with the “recenter particles using alignments” option checked. I tried running extraction at several points after sym expansion with that option unchecked, and all boxes were centered on the center of the entire capsid, as expected.
Sym expansion is indeed before vol alignment, the particles input to the Sym Expansion job are the direct output of a homo refine job with I symmetry applied. Here is a homo reconstruct only (C1) job run with the output of the Remove Duplicates job described in step 4 of my protocol:
So the center has been moved away from the capsid center. Since the shifts used in the Vol Align Tools job were calculated from a centered capsid, it’s not surprising they result in misaligned particles when applied to the particles that reconstruct the volume in the image above. In retrospect, my Remove Duplicates job in step 4 would not have worked unless the Sym Expand job moved the centers, so I should have planned for that, but I don’t see any indication that recentering occurs in the CS guide page for sym expansion, nor does such a shift seem to be taken into account in the protocol I linked to earlier. They don’t Remove Duplicates so could that somehow introduce the shift? It seems very unlikely to me.
I’m going to re-measure the coordinates to use for recentering in the Vol Align Tools job from the homo recontruct only map I posted in this comment, which I expect will resolve the issues with subtraction, re extraction, etc. However I would like to know for sure the source of the ‘extra’ center offset I’m observing, and how to predict or adjust its magnitude.