A cryo-EM grid preparation device for time-resolved structural studies

Dimitrios Kontziampasis, David P. Klebl, Matthew G. Iadanza, Charlotte A. Scarff, Florian Kopf, Frank Sobott, Diana C. F. Monteiro, Martin Trebbin, Stephen P. Muench (Lead / Corresponding author), Howard D. White (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

65 Citations (Scopus)
38 Downloads (Pure)

Abstract

Structural biology generally provides static snapshots of protein conformations that can provide information on the functional mechanisms of biological systems. Time-resolved structural biology provides a means to visualize, at near-atomic resolution, the dynamic conformational changes that macromolecules undergo as they function. X-ray free-electron-laser technology has provided a powerful tool to study enzyme mechanisms at atomic resolution, typically in the femtosecond to picosecond timeframe. Complementary to this, recent advances in the resolution obtainable by electron microscopy and the broad range of samples that can be studied make it ideally suited to time-resolved approaches in the microsecond to millisecond timeframe to study large loop and domain motions in biomolecules. Here we describe a cryo-EM grid preparation device that permits rapid mixing, voltage-assisted spraying and vitrification of samples. It is shown that the device produces grids of sufficient ice quality to enable data collection from single grids that results in a sub-4Å reconstruction. Rapid mixing can be achieved by blot-and-spray or mix-and-spray approaches with a delay of ∼10ms, providing greater temporal resolution than previously reported mix-and-spray approaches.

Original languageEnglish
Pages (from-to)1024-1031
Number of pages8
JournalIUCrJ
Volume6
Issue numberPart 6
DOIs
Publication statusPublished - 1 Nov 2019

Keywords

  • microscope hardware
  • sample preparation
  • structural biology
  • time-resolved cryo-EM
  • voltage-assisted spraying

ASJC Scopus subject areas

  • General Chemistry
  • Biochemistry
  • General Materials Science
  • Condensed Matter Physics

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  • A cryo-EM grid preparation device for time-resolved structural studies

    Kontziampasis, D., Klebl, D. P., Iadanza, M. G., Scarff, C. A., Kopf, F., Sobott, F., Monteiro, D. C. F., Trebbin, M., Muench, S. P. (Lead / Corresponding author) & White, H. D. (Lead / Corresponding author), 4 Mar 2019, BioRxiv, p. 1-22, 22 p.

    Research output: Working paper/PreprintPreprint

    Open Access

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