Membrane protein-micelle density

3D Refinement
1

I am new in the cryoSparc program and slowly learn the software to treat the membrane protein datasets. Thanks so much for your suggestions and ideals in advance. I have one membrane protein (38KDa) with Fab complex. so we have be able to pickup the particle including Fab complex but at the same time we throw_away a lot of particles for micelle without Fab. Some particle have bad Fab and micelle connection, which we also throw away. now the resolution is around 4.7-5A after refinement but the helix-TM isn’t good and a lot of micelle density.

questions: 1) how can I remove those micelle density so that I can see the helix-TM density clear?
2) since I already know the Antibody structure and TM structure, I don’t care about Fab structure how to interact with TM-micelle. Can I only try to pickup the TM-micelle particle (not pickup the TM-micelle with Fab) so that I can keep more particles? How can I do that (mask specific area of TM-micelle)?
3) if I knew the Antibody structure and TM structure, is the way that I can use this model to improve the cryo-EM density, which is very model bias, but I just want to quickly see the density and model fit.

2

Hi @jwangliang,

Apologies for the delay in my reply. These are interesting questions – what have you tried since? Is the entire structure 38 kDa? With structures that small, it is quite challenging in general to refine to high resolution in most cases.

In general with small proteins, it could also be challenging to tune particle picking to only pick up the TM/micelle region and ignore the fab. One way this could be done, though, is by iterating the refinement and particle picking stages. If you have a starting structure of the TM/micelle region without the fab, you can try the Create Templates job and then use these templates for Template Picking, which could help to pick specifically particles that exclude the Fab.

Best,
Michael

1 Like
3

Thanks so much Michael.
It would be interesting to test that. I will explore those option to see how to improve the density.
Jing :slight_smile: