Hi,
I would like to be able to explain why we consistently get these broad dips between 5.3Å and 7.0Å. Any help would be appreciated - happy to provide other info. The attached image was collected from a NU-Refinement job.
Brian
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Likely because one of the regions of your density is much less ordered than the rest (ex. detergent micelle, or a relatively mobile domain). You can try the 3D-VA job type (or cryoDRGN/ multi-body refinement in Relion) to get a better idea of if/where there is motion. Once you’ve identified the motion, you can run local refinement.
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I believe your suggestion is likely correct. We can in fact point to the regions with a lot of mobility, but have had limited success with local refinement. I am glad I can link this phenomenon to something we can rationalize and explain biologically. Thank you for your suggestions!
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What does the particles orientation look like? Is there any orientation bias?
There isn’t really. The “particles” are actually filamentous and wind around in a broad helix of different sizes and shapes. We used a blob picker to pick along the filaments as the filament tracer does not work well for heterogenous filaments, but we get a good distribution.
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