Dear all I have a question about the 3D classification of a membrane protein using Relion 3.1, where I aim to remove some junk particles. The protein is relatively small (180 kDa), and its structure has already been resolved by cryoEM. It has minimal features outside the micelle ring and is primarily composed of an alpha-helical bundle. Instead of generating a 3D initial model, I’m considering using the solved structure to create a reference map for the subsequent 3D classification and refinement steps. Therefore would it be more appropriate to use its PDB model (molmap) or the deposited EM map to create the reference map? The EM map includes the micelle ring, which the reference map from the model obviously will not have. Could this difference affect the classification or refinement steps, particularly when the protein is small and the map is low-pass filtered to 60 Å (default)? Also can I modify the resolution limit of the low-pass filter to improve classification?
Additionally, the molecule has C2 symmetry. Would it be better to use a C2 symmetric map for classification, or should I opt for a C1 asymmetric map? In 2D classification, I set 'Ignore CTF until first peak' to 'Yes' and limited the E-step resolution to 10 Å. Should I apply the same settings for 3D classification, especially regarding the 'Ignore CTF until first peak' option, or would it be better to set this to 'No'? Lastly, when I set 'Ignore CTF until first peak' to 'Yes' during 2D classification, it resulted in better class distribution compared to 'No,' where all particles plus junk tended to cluster in one class. What could explain this behavior? Is it something specific to small membrane proteins with fewer features? Your advice would be greatly appreciated. Best Firdous ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
