2020 Volume 55 Issue 1 Pages 37-42
Recently, it has been demonstrated that single particle analysis using 200 kV electron cryo-microscope is capable of reconstructing protein structures at resolution higher than 3.0 Å. Because of the enhanced contrast of particle images taken by 200 kV microscope compared with 300 kV, proteins smaller than 100 kDa were also reconstructed at the same resolution level without using large defocus or phase plate. This is, the 200 kV should be more suitable for small proteins (< 200 kDa). However, the majority of near-atomic resolution cryo-EM structures has been determined using 300 kV. As a consequence, many of typical parameter settings for imaging session and image processing steps, especially ones associated with the contrast transfer function, are based on the accumulated experience of the higher acceleration voltage. Here, we will revise these parameters for 200 kV, establish theoretical base for criteria to find an optimal box size and particle mask diameter for a given dataset, and explain a proposed protocol. Examples of actual analysis are also given. In these, by considering the defocus distributions, merely optimizing the box sizes and particle mask diameters yielded prominent improvements from resolutions around 3.0 Å in the reconstructions of < 200 kDa proteins.
