I have collected relatively good data on my disk-shaped, trimeric the protein, but I cannot get good 3D reconstruction and I cannot figure out why. My classes look good (see attached), I think, and there are over 30,000 particles chosen. I have run many 3D classifications to clean out any bad particles and I think it is a relatively clean picking.
Ab initio 3D gives me very poor results (see attached), appearing like it simply cannot properly overlay the particles in a reasonable way. Also, when I try to refine against a 20-angstrom map of the known structure of a nearly identical protein, the resolution never goes beyond 14 angstroms, and the slices only get worse from the start.
I have tried making the refinement package in C1 and C3. I have tried different size/location parameters in the Expert Settings in both Ab initio and AutoRefinement. I have tried to add a class in the refinement package and it gives me ~1% for the second particle, so I assume that means there is really only 1.
I am really out of ideas and I cannot seem to make progress. If anyone has any input, I would appreciate it.
Here is some extra information on the shape and size of the protein: The protein has C3 symmetry so that each wedge (monomer) of the disk are equivalent. On one side of the disk, there is a bump on each of the three wedges. The other side is pretty flat If considered a disk, the protein is about 300 angstroms in diameter and the thickness is ~40 angstroms, not including the bump region on the one side which would add another ~15-20 angstroms.