Hi,
Novice cryosparc user here. In my 3D refinement workflow, I obtain a 4.6A map after homogenous refinement (C1 symmetry/Do symmetry alignment checked green). The map looks promising (although as expected some regions are better than the other). When I open the map on chimera, the Z axis is along perpendicular to screen (that is the map gets displayed along the Z axis first as I open it on chimera). The molecule (which is a dimer) and the map looks like it has C2 symmetry although parts of one dimer is better resolved that the similar parts in the other.
I took the particles/volume/mask from this homogenous refinement and ran another homogenous refinement with identical parameters but with C2 symmetry. The newly generated map looks terrible and looks almost like featureless blob with random blobs jutting out. (The new “meaningless map” looks like is symmetric about the Y axis)
If instead of the previous step, I take the particles/volume/mask from the first homogenous refinement job and run local refinement (with the Non-uniform refinement option checked green) then I get a 4.15A map (and the map quality is an improvement). The C2 symmetry in the molecule is even more apparent in this map (although there are clear differences in the quality of the maps in one of the dimers vs the other.
I cannot seem to figure out why imposing symmetry makes the map so much worse - makes me think that I am doing something wrong but don’t know what. (On chimera when I use: measure center #map I get a set of non-zero coordinates. I don’t know if that is the problem? As in does the center of the map need to fall on the Z axis aka z coordinate of the center need to be 0? If so how can I do this? Or does the symmetry axis of the molecule need to be the Y axis and if so how can I change the reference map to have its symmetry axis along the Y axis)
Any help would be appreciated.