I have a volume that I’m almost finished refining, but it’s left-handed so I need to flip it. Would you recommend finishing refinement with the original left-handed map, then flipping right at the end? I plan to do a little local refinement but it shouldn’t need much else at this point. I’m concerned that flipping the volume now will alter how the individual particles are handled (I would assume that some feature of the particles is causing this handedness – what would this be if they’re just projections?) Alternatively, is there a benefit to flipping during refinement?
Hi,
You could try using Homogeneous Reconstruct Only, which has the option of “Flip the reconstruction hand”.This will flip both the map and the particles. Then you can proceed with Local Refinement.
The problem with the handedness comes from the nature of a 2D projection. A top view looks the same as a a bottom view. This is true for all projections and their 180° counterpart. Therefore, the handedness cannot be determined during back projection. Consequently, 50% of your ab-initios and all subsequent refinement will have the wrong handedness.
You can flip the reference volume, which can be your already refined map, and use it for another consensus refinement. There are no differences in reconstruction quality. Just make sure to refine the particles against the fliped map before doing any masking/local refinements.
You can do this at any point in yor workflow, even after the final (local) refinement(s). However, it makes sense to flip it as soon as possible in order to work on your model, compare other published maps etc wothout the need of flipping it first.
Tip: If you have a high enough resolution, you can see if your handedness is correct on the alpha-helices. If it is correct, they will have a right handed coil.