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

David P. Klebl; Molly S. C. Gravett; Dimitrios Kontziampasis; David J. Wright; Robin S. Bon; Diana C. F. Monteiro; Martin Trebbin; Frank Sobott; Howard D. White; Michele C. Darrow; Rebecca F. Thompson; Stephen P. Muench

doi:10.2139/ssrn.3608388

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

David P. Klebl, Molly S. C. Gravett, Dimitrios Kontziampasis, David J. Wright, Robin S. Bon, Diana C. F. Monteiro, Martin Trebbin, Frank Sobott, Howard D. White, Michele C. Darrow, Rebecca F. Thompson (Lead / Corresponding author), Stephen P. Muench (Lead / Corresponding author)

Mechanical and Industrial Engineering

Research output: Working paper/Preprint › Preprint

Abstract

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.

Original language	English
Publisher	Social Science Research Network
Pages	1-41
Number of pages	41
Volume	28
DOIs	https://doi.org/10.2139/ssrn.3608388
Publication status	Published - 16 Jun 2020

Keywords

air-water-interface
cryoEM
sample preparation

ASJC Scopus subject areas

Structural Biology
Molecular Biology

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10.2139/ssrn.3608388

Cite this

Klebl, D. P., Gravett, M. S. C., Kontziampasis, D., Wright, D. J., Bon, R. S., Monteiro, D. C. F., Trebbin, M., Sobott, F., White, H. D., Darrow, M. C., Thompson, R. F., & Muench, S. P. (2020). Need for Speed: Examining Protein Behavior during CryoEM Grid Preparation at Different Timescales. (11 ed.) (pp. 1-41). Social Science Research Network . https://doi.org/10.2139/ssrn.3608388

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abstract = "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{\texttrademark}, 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 {\textquoteleft}silver bullet{\textquoteright} for improving the quality of every cryoEM sample preparation.",

keywords = "air-water-interface, cryoEM, sample preparation",

author = "Klebl, {David P.} and Gravett, {Molly S. C.} and Dimitrios Kontziampasis and Wright, {David J.} and Bon, {Robin S.} and Monteiro, {Diana C. F.} and Martin Trebbin and Frank Sobott and White, {Howard D.} and Darrow, {Michele C.} and Thompson, {Rebecca F.} and Muench, {Stephen P.}",

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doi = "10.2139/ssrn.3608388",

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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.

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

Abstract

Keywords

ASJC Scopus subject areas

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

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

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