Need for speed: Examining protein behaviour during cryoEM grid preparation at different timescales

TY - UNPB

T1 - Need for speed

T2 - Examining protein behaviour during cryoEM grid preparation at different timescales

AU - Klebl, David P.

AU - Gravett, Molly S. C.

AU - Kontziampasis, Dimitrios

AU - Wright, David J.

AU - Bon, Robin S.

AU - Monteiro, Diana C. F.

AU - Trebbin, Martin

AU - Sobott, Frank

AU - White, Howard D.

AU - Darrow, Michele C.

AU - Thompson, Rebecca F.

AU - Muench, Stephen P.

PY - 2020/5/15

Y1 - 2020/5/15

N2 - 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.

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.

UR - https://www.scopus.com/pages/publications/85090243631

U2 - 10.1101/2020.05.14.095372

DO - 10.1101/2020.05.14.095372

M3 - Preprint

SP - 1

EP - 33

BT - Need for speed

PB - BioRxiv

ER -