2D classification to show satellite particles

Hi everyone,

I’m working with a dataset that contain a main particle (70 kDa) bound to two satellite particles (30 kDa each). Using ‘standard’ processing, I was able to solve the structure of the main particle at 3.5A. The satellite particles are not-visible in any step of the processing, but can be seem in the extracted particles

I would like to show that they exist at least in 2D classification. For that, I extracted the particles using larger boxes and tried to 2DC those modifying parameters like Max res, solvent clamp, uncertainty factor, etc. The results I have so far are not convincing

any suggestion of how I configure my 2DC for enhance visualization of these particles?


Hi, have you tried signal subtraction of your main particle? You could take a multi-body refinement approach, but going after a 30kda sub-particle might be difficult (although definitely worth a shot!).
Alternatively, can you get the satellite particles to show up in any 3D methods? Maybe ab initio into multiple classes without going to high resolution could help detect them? I feel like significantly limiting the resolution through binning could help by preventing alignment based on the high resolution features and force the algorithms to only classify based on low res features.

Hi @user123 Could you please tell how to do signal subtraction in CryoSparc or relion? Thanks a lot?

Until other users chime in with their practical experiences, you may find some relevant information in the guide.

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