EMD-29688

Single-particle
3.2 Å
EMD-29688 Deposition: 06/02/2023
Map released: 06/12/2023
Last modified: 14/02/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links

EMD-29688

Time-resolved cryo-EM study of the 70S recycling by the HflX:1st intermediate

EMD-29688

Single-particle
3.2 Å
EMD-29688 Deposition: 06/02/2023
Map released: 06/12/2023
Last modified: 14/02/2024
Overview 3D View Sample Experiment Validation Volume Browser Additional data Links
Sample Organism: Escherichia coli
Sample: Apo 70S 1st intermediate
Fitted models: 8g34 (Avg. Q-score: 0.116)

Deposition Authors: Bhattacharjee S , Brown PZ, Frank J
Time resolution in cryo-EM using a PDMS-based microfluidic chip assembly and its application to the study of HflX-mediated ribosome recycling.
Bhattacharjee S , Feng X, Maji S, Dadhwal P, Zhang Z, Brown ZP, Frank J
(2024) Cell , 187 , 782 - 796.e23
PUBMED: 38244547
DOI: doi:10.1016/j.cell.2023.12.027
ISSN: 1097-4172
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 1,000 ms, the device allows us to follow in vitro reactions of biological molecules at resolution levels in the range of 3 Å. 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 140 ms.