Thanks for the reply 
I have since done a lot of processing in cryosparc and it is amazing to see how the results have improved!
You asked if the dimer top is definitely dimer top and not two monomers closely spaced - I can’t really say for sure. That has been the crux of my issue with this sample. It is too small to see on the micrographs and is only visible by eye after Topaz denoising. For that reason, I previously relied on 2D classification but I suspect that it was influenced by the attraction problem as different conformations or monomer/dimer were often pooled together. I iteratively separated them out but the SNR ultimately decreased and may have resulted in true particles being discarded.
Running the whole stack through cryosparc ab initio has helped and the results are much better now that human bias was reduced. It is still difficult, however, to choose a value for K because of the particle’s shape. Each domain is a sphere at low resolution and CS has pooled dimer side with monomer side (since what is visible of the dimer is basically a monomer), dimer front view with monomer side (not sure why) and dimer bottom with monomer side (both are composed of 2 domains although the spacing of dimer bottom is closer while the monomer has a linker in-between).
Other complicating factors are that
- each domain undergoes hinging to open and close the active site cleft
- there is flexibility at the inter-domain linker
- there is flexibility in the glycans on the surface
I can solve a good structure using NU-refine when the cleft is visible but cannot when the cleft is closed (both domains are just spheres with a slightly extended flexible linker and glycans on the surface). I think that this flexibility may impede protein alignment, even with NU-refinement. There’s a lot of blurry signal on the surface and even if I extend the dynamic mask to 12-18A and use a threshold of 0.1, I still get a sharp drop in corrected FSC. Am I doing something wrong?
