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bioRxiv · 10.1101/2022.10.21.513149

Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting

TORINO, S.·Dhurandhar, M.·Stroobants, A.·Claessens, R.·Efremov, R. G.
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Abstract

Using single particle cryogenic electron microscopy (cryo-EM) high-resolution structures of proteins in different conformations can be reconstructed. 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 initiation of reaction and rapid 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 spatial resolution of 2.1 [A]. We performed trEM experiments on GroEL:GroES chaperonin complex, these resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex.

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TORINO, S., Dhurandhar, M., Stroobants, A., Claessens, R., Efremov, R. G.. 2022-10-21. Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting. https://doi.org/10.1101/2022.10.21.513149

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