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)

Research output: Contribution to journalArticlepeer-review

46 Citations (Scopus)
16 Downloads (Pure)


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.

Original languageEnglish
Pages (from-to)1238-1248.e4
Number of pages16
Issue number11
Early online date18 Aug 2020
Publication statusPublished - 3 Nov 2020


  • air-water-interface
  • cryoEM
  • HSPD1
  • ribosome
  • sample preparation

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology


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