When I turn on ‘Min over scale after first iteration’ during 2D classfication, I get two different looking 2Dclasses, one light and one dark. Is the dark 2D class a ‘bad’ class? Should I keep the dark 2D classes especially if it seems to have high resolution features?
I have never seen this so I’m not exactly sure what is going on here - my first guess would be that the scale refinement might be unstable, and for these particles it could be minimizing to a negative value (explaining the sign inversion).
Probably the easiest way to tell would be to select this class and have a look at the raw particles on the micrographs using inspect particle picks - do they look unusual in any way?
Thank you for your comment. I selected the dark 2D classes and inspected the particles, but, overall, particles seem fine. It is avoiding ice contamination. There are very few wrong particle picks here and there, but I would assume that is the case for all 2D classes.
Is it safe to assume that these dark classes will not be negatively affect the 3D reconstruction?
When I inspected the micrographs, the contrast was not inverted (dark particles on light background). Also, the data collection was done between -1 um and -2.5 um defocus, which was more or less consistent based on the estimated defocus value from CTF estimation. Based on these, I do not think they are overfocused.
Dark classes do not appear with the normal 2D classifications. However, when I compared results from with and without minimizing scale factors, it seems like I get better junk separation. Below is the 2D class results from without minimizing scale factors.