Stuck at ~4 Å after HR ab initio and local CTF refinement

Cryo-EM Data Processing
1

Hi,

I am working on a membrane protein dataset and trying to further improve its resolution in cryoSPARC.

So far, I processed the data through heterogeneous refinement to separate particles into distinct structural classes, iteratively removing junk and improving class quality until reaching a stable and clean dataset. This resulted in an improved map at around 4 Å resolution.

From there, I took the selected particles into high-resolution ab initio reconstruction using two classes. I continued processing with only one of these classes (in the other class, one dynamic subunit was missing) and performed local CTF refinement, which resulted in a map at approximately 4.1 Å.

At this point, I am unsure how to further improve the resolution within cryoSPARC. I would appreciate recommendations on which jobs or strategies to apply next to push the resolution higher.

Thanks !!

2

hi shalu,

Can you give us a bit more information here? how many particles do you have? how big is your protein? is it in detergent and how are you masking? have you done a standard local refine job (not CTF refine) following the ab-initio? does doing homo refine or NU-refine make your resolution worse? do you have areas of clearly better and worse local resolution in your reconstruction, and does most of it actually look like 4A or is that clearly an overestimate (e.g. can you see helices? side chains?).

lots of questions but hard to suggest what to do next without understanding where you are at now. it’s always possible that you are at the maximum resolution for your data set, especially if it’s a small membrane protein. what resolution do you need to get to in order to answer the biological questions that you are trying to ask?