Hi everyone,
I am working on the reconstruction of a microtubule-like filament. There appears to be a small protein attached to the outer surface of the filament, and this peripheral protein may be quite flexible.
Before proceeding to Helical Refinement, I would like to ask what aspects of the upstream processing should be handled especially carefully in order to preserve this density as much as possible. For example, are there any particular considerations in particle picking, extraction, 2D classification, initial model generation, mask design, symmetry choice, or particle selection that are especially important when a flexible peripheral component is present?
In addition, I would also like to ask how to evaluate whether the final reconstruction is correct and reliable in this situation. If the peripheral density becomes weak or disappears after Helical Refinement, how can I tell whether this is mainly due to flexibility, low occupancy, incorrect symmetry, alignment bias toward the filament core, or some issue in the upstream processing?
Any suggestions, practical strategies, or recommended workflows would be greatly appreciated. Thank you very much for your help.