Hi @olibclarke, thank you again for the suggestions. I appreciate that you have a lot on your plate, helping others on the forum too, so all your advice really is appreciated.
I tried homogenous reconstruction on all 3 classes separately, and it looks like the dimer shows up well. I noticed a 6.5 Å “resolution bump” as described elsewhere. I wonder if this is because I added detergent to my sample or if the protein complex is flexible like that. (I added detergent to reduce orientation bias, and there was too much protein tightly packed on the grid in initial screening endeavors.) In any case, it looks like I can see some overall architecture of the protein complex, especially the dimer.
Local refinement is less than ideal currently. You can see that even when I locally refine to the “tight dimer,” I am getting pretty bad deterioration of the GFSC curves. I’m showing one masking strategy and the subsequent volume outputs to demonstrate what the actual maps look like after refinement. Happy to show the other ones too, though I’m not sure if it’ll be too helpful. My concern is that the monomer isn’t there, but it’s strange that the density is much more prominent in Relion. Hopefully, I’m missing something in the local refinement runs and this isn’t some inherent limitation of the dataset. I’ve also tried 3D classification (without alignment) jobs and “Ab Initio” jobs in Relion, and the density does seem to be there and not artifactual, though my interpretation may be due to less mature understanding of how alignment algorithms work in both of these programs. To demonstrate what I mean, I’m attaching an image of a Relion 2D classification “diagnostic” run. (These particles underwent several rounds of 2D/3D classification.) I will give HR-HAIR with start/final resolution at 5-6 Å next. Could this possibly be limiting the monomer density? (i.e. HR-HAIR fits well to the dimer, but the monomer takes a hit because the alignment is on these finer/higher-res features?)
Multiple rounds of 2D/3D classification (Relion, please ignore red outlines/picks, since these weren’t the particles I imported into CryoSPARC):
I also ran a 3D classification job on one of the homogenous reconstructions (66,737 particles) with a focus map in the monomer density region (filter resolution = 10 Å), pulling out some possible monomer density…
Finally, if it might help diagnose, here are the local refinement alignment changes (1 is the tight dimer, 2 is the ? monomer). It seems like the “monomer local refinement” is not working too well with massive drift, despite the GFSC measure. Perhaps this is starting to get outside of the scope of the initial question, so I might make a new post…
Thanks again so much for the wisdom and help!