8BKG

Cryo-EM structure of beta-galactosidase at 3.2 A resolution plunged 35 ms after mixing with apoferritin

Summary for 8BKG

• Entry DOI: 10.2210/pdb8bkg/pdb
• EMDB information: 16097
• Descriptor: Beta-galactosidase (1 entity in total)
• Functional Keywords: glycosyl hydrolase, hydrolase
• Biological source: Escherichia coli K-12
• Total number of polymer chains: 4
• Total formula weight: 469953.50

Authors

Torino, S.,Dhurandhar, M.,Efremov, R. (deposition date: 2022-11-09, release date: 2023-08-09, Last modification date: 2023-09-13)

Primary citation

Torino, S.,Dhurandhar, M.,Stroobants, A.,Claessens, R.,Efremov, R.G.
Time-resolved cryo-EM using a combination of droplet microfluidics with on-demand jetting.
Nat.Methods, 20:1400-1408, 2023

PubMed Abstract: Single-particle cryogenic electron microscopy (cryo-EM) allows reconstruction of high-resolution structures of proteins in different conformations. 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 reaction initiation and 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 resolution of 2.1 Å. trEM experiments on GroEL:GroES chaperonin complex resolved the kinetics of the complex formation and visualized putative short-lived conformations of GroEL-ATP complex.

PubMed: 37592181
DOI: 10.1038/s41592-023-01967-z

Experimental method

ELECTRON MICROSCOPY (3.2 Å)