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
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
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.
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!
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.