I am using cryosparc 3.2.0 to get the structure of a protein complex (composed of two different proteins) and has difficulties to make sense of the map I got, although the resolution looks quite promising.
I initially selected around 875,000 particles from 2D classification, then an ab initio run with 2 classes, following a hetero refinement and I then run refinement with the best looking class (around 640,000 particles) which gave a map around 3.0A (applying C1 symmetry). However, the map looks a bit odd from one side (It has a sort of bump). Considering the class of this protein family, I expect a symmetric solution. Indeed, when I look at some of the images from 2D classification, I could really count obvious corners. I also tried to impose symmetry (such as C3, C6, D6) during hetero refinement and refinement steps, yet it resulted in worse resolution (>5.5A). In addition, I run ab initio job using the best class above (640k) and tried to divide into more classes (3 different) and run hetero refinement, but the solutions I got is more or less the same. Finally, I also picked less amount of particles from 2D classification which looked quite clear, but again one side the complex looks a bit unexpected to me
I am relatively new to cryo-em. So, I will highly appreciate if anyone could give any insights what might be wrong or what I am missing. Perhaps,
Not enough views (info) for this side of the complex, so couldn’t construct that part?
Symmetry problem? (I highly would doubt, I tried to impose all the available symmetry options)
Has to tune ab inito reconstruction parameters? (I run several runs by changing initial and final minibatch size (increasing)) and initial learning rate.)
Perhaps, it is what it is!
Thank you very much!