I’m processing a three-subunit complex with an expected molecular weight of about 120 kDa. I collected 19,238 micrographs on a Titan Krios and have about 2 million particles. The CTF estimates look very good, and the 2D class averages look promising.
At the heterogeneous refinement stage, the resolution is about 9.5 Å. Non-Uniform Refinement makes it slightly worse, to about 9.6 Å. I see two apparent orientations of one subunit relative to the other two, and I’d like to improve the resolution of both states.
It is difficult to provide informed suggestions without seeing the 2D classes, FSCs etc - can you provide these? E.g. in the 2D classes do you see clear secondary structure? Do your ab initio volumes have a molecular envelope that matches your expectations based on e.g. alphafold or comparable structures?
Thanks, Oliver. I’ve attached some 2D class averages, the FSC plot from Heterogeneous Refinement for the full-complex class, and the corresponding FSC from NU Refinement.
Yes, the ab initio volumes have an overall molecular envelope consistent with our expectations based on a related experimentally resolved structure.
Interestingly, the two volumes appear to show the third unit connected to different units of the core.
From your 2D averages i assume that each of the small subunit is ~40 kDa of your 120kDa complex. Hence its too small. You have 2 million particles which is great. I would first clean up until i see a bit clean features. From you subunits it seems that there is also flexibility in your subunits. So do a cleanup removing as many junk as possible and then use ab initio high res cut off say 7 to 10 for initial 2-3 classes. And the take the best possible class for Homogeneous ab initio refinement (cryosparc ver 5.0 onwards) or HR HAIR protocol by @olibclarke. I guess it should help.
