Smeared 2D classification?

[niu]

Hi, all

I had a protein with a molecular weight of 120-kDa. We collected a dataset on K3 camera with the raw images that look good. However, the 2D-classfication results display a smeared pattern after a series of the general processes. I am not sure if the particles locate in the air-water interface or some other mistake happened? Dose someone meet the similar trouble or give any suggestion?

thanks,

niu

[CryoEM1]

Your 2D look great. There is some flexibility between the two domains (proteins). You should use a slightly smaller box maybe 80% as large, at least until highest resolution reconstructions.