I am working on a EM map which NU refinement with 66K particles results in 3.6A (C1 symmetry) and 2.85A (C11 symmetry). The C1 map shows DNA molecle attached to the 11-mer ring but C11 map doesn’t show DNA molecule attached to the 11-mer ring. During the 3DVA I am doing symmetry expansion job and if I apply C11 smmetry the DNA density map will disappear. Any thoughts about running 3DVA job and resolving the affect of DNA on the 11-mer ring? Or any other methods to check the hetergeneity of the protein-DNA complex?
Thank you,
Marty,
Hi Marty, I would recommend performing symmetry expansion after your C11 refinement, then 3D classification without alignments in cryosparc, with no mask input (or a mask that is broad enough to include the ring and the DNA binding sites supplied as solvent mask).
A target resolution of 15Å will probably be sufficient to resolve binding/unbinding. You will want to include enough classes to allow for bound/unbound states at each position, so maybe 30-50 classes? Test with and without force hard classification.
Then you can combine the appropriate classes, rotate and/or deduplicate if necessary, then proceed to local refinement in C1.
Cheers
Oli
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Hi Oli,
Thank you so much for your suggestions. I was wondering how can I combine multiple classes from 3D classification job into one and run local refinement?
Thank you,
Marty
Rotate classes if needed using Volume alignment tools, then combine outputs in a single homogeneous reconstruction job. Use the particle & volume outputs from homogeneous reconstruction to start a local refinement job.