Large protein complex with symmetric part and asymmetric part

Cryo-EM Data Processing
1

Hi, all.

Currently I’m working with a protein complex with molecular weight greater than 1.5 kDa. This complex is composed of two rigid parts, one with D6 symmetry and is exposed to the solvent region, the other does not have any symmetry and is embbed in the micelle.
Main problem with this complex is that solvent part can be resolved clearly with high resolution, but the membrane-embedded part is not well resolved. I tried NU refinement with C1 symmetry, hoping all particles can find their right angles in the volume, but this just dosen’t work. And I also tried to use mask covering different range of the volume, but the results were also not good.
I am wondering if there is any strategies can be further applied for refinement or classification?

2

A couple of things I would try:

  • specifically picking the micelle region, tight mask for 2D to get micelle region centred and focussed, then try reconstructing
  • D6 reconstruction of the whole thing, symmetry expansion followed by 3D classification and/or local refinement and recentring on a custom mask of where the micelle region should be

Given the whole complex is ~1.5MDa, what is the approximate size of the membrane region vs. the whole complex?

Welcome to the forum. :slight_smile:

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