PT  - JOURNAL ARTICLE
AU  - David P. Klebl
AU  - Molly S. C. Gravett
AU  - Dimitrios Kontziampasis
AU  - David J. Wright
AU  - Robin S. Bon
AU  - Diana Monteiro
AU  - Martin Trebbin
AU  - Frank Sobott
AU  - Howard D. White
AU  - Michele Darrow
AU  - Rebecca F. Thompson
AU  - Stephen P. Muench
TI  - Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales
AID  - 10.1101/2020.05.14.095372
DP  - 2020 Jan 01
TA  - bioRxiv
PG  - 2020.05.14.095372
4099  - http://biorxiv.org/content/early/2020/05/15/2020.05.14.095372.short
4100  - http://biorxiv.org/content/early/2020/05/15/2020.05.14.095372.full
AB  - A host of new technologies are under development to improve the quality and reproducibility of 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 behaviour 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 optimise. 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.Competing Interest StatementThe authors would like to acknowledge that MCD works for SPT Labtech, the company developing and manufacturing chameleon systems.