Size matters: optimal mask diameter and box size for single-particle cryogenic electron microscopy

Recently it has been demonstrated that single-particle cryogenic electron microscopy (cryo-EM) at 200 keV is capable of determining protein structures, including those smaller than 100 kDa, at sub-3.0 Å resolutions, without using significant defocus or a phase plate. However, the majority of near-atomic resolution cryo-EM structures has been determined using 300 keV. Consequently, many typical parameter settings for the cryo-EM computational image processing steps, especially those associated with the contrast transfer function, are based on the accumulated experience of 300 kV cryo-EM. We have therefore revised these parameters, established theoretical bases for criteria to find an optimal mask diameter and box size for a given dataset irrespective of acceleration voltage or protein size, and proposed a protocol. Considering the defocus distributions of the datasets, merely optimizing the mask diameters and box sizes yielded meaningful resolution improvements for the reconstruction of < 200 kDa proteins using 200 kV cryo-EM.