Samenvatting

Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. Protein function often involves transient functional conformations, which can be resolved using time-resolved cryo-EM (trEM). In trEM, reactions are arrested after a defined delay time by rapid vitrification of protein solution on the EM grid. Despite the increasing interest in trEM among the cryo-EM community, making trEM samples with a time resolution below 100 ms remains challenging. Here we report the design and the realization of a time-resolved cryo-plunger that combines a droplet-based microfluidic mixer with a laser-induced generator of microjets that allows rapid reaction initiation and plunge-freezing of cryo-EM grids. Using this approach, a time resolution of 5 ms was achieved and the protein density map was reconstructed to a resolution of 2.1 Å. trEM experiments on GroEL:GroES chaperonin complex resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL–ATP complex.

Originele taal-2English
Pagina's (van-tot)1400-1408
Aantal pagina's9
TijdschriftNature Methods
Volume20
Nummer van het tijdschrift9
DOI's
StatusPublished - sep 2023

Bibliografische nota

Funding Information:
We thank H. De Greve for designing primers used to clone the lacZ gene. We are indebted to A. Manz (KIST, Saarbrüken) for helpful discussions, B. Schied (ULB, Brussels) for a practical introduction to microfluidics and help with setting up microfabrication laboratory, and M. Fislage for assistance with cryo-EM data collection. We would like to acknowledge the funding provided by Vlaams Instituut Voor Biotechnologie, Fonds Wetenschappelijk Onderzoek (grant nos. G0H5916N, G054617N to R.G.E.), and by the European Research Council (grant no. 726436 to R.G.E.). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Publisher Copyright:
© 2023, The Author(s), under exclusive licence to Springer Nature America, Inc.

Copyright:
Copyright 2023 Elsevier B.V., All rights reserved.

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