RT Journal Article
SR Electronic
T1 Visual Biochemistry: modular microfluidics enables kinetic insight from time-resolved cryo-EM
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 2020.03.04.972604
DO 10.1101/2020.03.04.972604
A1 Märt-Erik Mäeots
A1 Byungjin Lee
A1 Andrea Nans
A1 Seung-Geun Jeong
A1 Mohammad M. N. Esfahani
A1 Daniel J. Smith
A1 Chang-Soo Lee
A1 Sung Sik Lee
A1 Matthias Peter
A1 Radoslav I. Enchev
YR 2020
UL http://biorxiv.org/content/early/2020/03/05/2020.03.04.972604.abstract
AB Mechanistic understanding of biochemical reactions requires structural and kinetic characterization of the underlying chemical processes. However, no single experimental technique can provide this information in a broadly applicable manner and thus structural studies of static macromolecules are often complemented by biophysical analysis. Moreover, the common strategy of utilizing mutants or crosslinking probes to stabilize otherwise short-lived reaction intermediates is prone to trapping off-pathway artefacts and precludes determining the order of molecular events. To overcome these limitations and allow visualisation of biochemical processes at near-atomic spatial resolution and millisecond time scales, we developed a time-resolved sample preparation method for cryo-electron microscopy (trEM). We integrated a modular microfluidic device, featuring a 3D-mixing unit and a delay line of variable length, with a gas-assisted nozzle and motorised plunge-freeze set-up that enables automated, fast, and blot-free sample vitrification. This sample preparation not only preserves high-resolution structural detail but also substantially improves protein distribution across the vitreous ice. We validated the method by examining the formation of RecA filaments on single-stranded DNA. We could reliably visualise reaction intermediates of early filament growth across three orders of magnitude on sub-second timescales. Quantification of the trEM data allowed us to characterize the kinetics of RecA filament growth. The trEM method reported here is versatile, easy to reproduce and thus readily adaptable to a broad spectrum of fundamental questions in biology.