Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling

Abstract

The rapid kinetics of biological processes and associated short-lived conformational changes pose a significant challenge in attempts to structurally visualize biomolecules during a reaction in real time. Conventionally, on-pathway intermediates have been trapped using chemical modifications or reduced temperature, giving limited insights. Here, we introduce a time-resolved cryo-EM method using a reusable PDMS-based microfluidic chip assembly with high reactant mixing efficiency. Coating of PDMS walls with SiO2 virtually eliminates non-specific sample adsorption and ensures maintenance of the stoichiometry of the reaction, rendering it highly reproducible. In an operating range from 10 to 1,000ms, the device allows us to follow invitro reactions of biological molecules at resolution levels in the range of 3Å. By employing this method, we show the mechanism of progressive HflX-mediated splitting of the 70S E.coli ribosome in the presence of the GTP via capture of three high-resolution reaction intermediates within 140ms.