Increasing vitrification temperature improves the cryo-electron microscopy reconstruction

Abstract

At the initial stage of the cryo-electron microcopy (cryo-EM) samples irradiated by electrons, the cryo-EM samples suffer from a rapid \"burst\" phase (first 3~4 e-/[A]2) of beam induced motion (BIM) which is too fast to be corrected by existing motion correction software, and lowers the quality of the initial frames. Therefore, these least radiation damaged, but ruined frames are commonly excluded or down-weighted during data processing, which reduces the undamaged signals in the reconstruction and decreases the reconstruction resolution by weakening the averaging power. Here, we show that increasing the freezing temperature of cryo-EM samples suppresses the BIM in this phase. The quality of initial frames is partially recovered after BIM correction and is better than that of subsequent frames in certain frames. Incorporating these initial frames into the reconstruction increases the resolution, at an equivalent of ~60% extra data. Moreover, these frames are least radiation damaged, thus preserves the high quality cryo-EM density of radiation sensitive residues. Such density is usually damaged or very weak in the canonical 3D reconstruction. In addition, we found that a different kind of radiation damage neglected previously occurs in the per-frame reconstruction after the exposure of 2.5 e-/[A]2. Such radiation damage distorts the density of atoms. The deformation can be avoided by only including the frames from the first 2.5 e-/[A]2 into the reconstruction. Overall, the high temperature freezing not only provides extra undamaged signal to the reconstruction, but also increases the resolution of the reconstruction.