PT  - JOURNAL ARTICLE
AU  - Torino, Stefania
AU  - Dhurandhar, Mugdha
AU  - Stroobants, Annelore
AU  - Claessens, Raf
AU  - Efremov, Rouslan G.
TI  - Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting
AID  - 10.1101/2022.10.21.513149
DP  - 2022 Jan 01
TA  - bioRxiv
PG  - 2022.10.21.513149
4099  - http://biorxiv.org/content/early/2022/10/21/2022.10.21.513149.short
4100  - http://biorxiv.org/content/early/2022/10/21/2022.10.21.513149.full
AB  - 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 Å. 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.Competing Interest StatementThe authors have declared no competing interest.