9mp8

Publication Abstract from PubMed

Time-resolved X-ray crystallography has great promise to illuminate structure-function relations and key steps of enzymatic reactions with atomic resolution. The dominant methods for chemically-initiated reactions require complex instrumentation at the X-ray beamline, significant effort to operate and maintain this instrumentation, and enormous numbers ( approximately 10(5)-10(9)) of crystals per time point. We describe instrumentation and methods that enable high-throughput time-resolved study of biomolecular systems using standard crystallography sample supports and mail-in X-ray data collection at standard high-throughput cryocrystallography synchrotron beamlines. The instrumentation allows rapid reaction initiation by mixing of crystals and substrate/ligand solution, rapid capture of structural states via thermal quenching with no pre-cooling perturbations, and yields time resolutions in the single-millisecond range, comparable to the best achieved by any non-photo-initiated method in both crystallography and cryo-electron microscopy. Our approach to reaction initiation has the advantages of simplicity, robustness, low cost, adaptability to diverse ligand solutions and small minimum volume requirements, making it well suited to routine laboratory use and to high-throughput screening. We report the detailed characterization of instrument performance, present structures of binding of N-acetylglucosamine to lysozyme at time points from 8 ms to 2 s determined using only one crystal per time point, and discuss additional improvements that will push time resolution toward 1 ms.

Instrumentation and methods for efficient time-resolved X-ray crystallography of biomolecular systems with sub-10 ms time resolution.,Indergaard JA, Mahmood K, Gabriel L, Zhong G, Lastovka A, McLeod MJ, Thorne RE IUCrJ. 2025 May 1;12(Pt 3):372-383. doi: 10.1107/S205225252500288X. PMID:40277177^[2]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.