Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales.

ABSTRACT: A host of new technologies are under development to improve the quality and reproducibility of cryoelectron microscopy (cryoEM) grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot, chameleon, and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms (the fastest reported to date), to interrogate particle behavior at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin-film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimize. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic "silver bullet" for improving the quality of every cryoEM sample preparation.

SUBMITTER: Klebl DP 

PROVIDER: S-EPMC7652391 | biostudies-literature | 2020 Nov

REPOSITORIES: biostudies-literature