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Título: | Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography |
Autor: | Pandey, Suraj; Calvey, George; Katz, Andrea M.; Malla, Tek Narsingh; Koua, Faisal H. M.; Martín-García, José M. CSIC ORCID; Poudyal, Ishwor; Yang, Jay-How; Vakili, Mohammad; Yefanov, Oleksandr; Zielinski, Kara A.; Bajt, Sasa; Awel, Salah; Doerner, Katarina; Frank, Matthias; Gelisio, Luca; Jernigan, Rebecca; Kirkwood, Henry; Kloos, Marco; Koliyadu, Jayanath; Mariani, Valerio; Miller, Mitchell D.; Mills, Grant; Nelson, Garrett; Olmos, Jose L.; Sadri, Alireza; Sato, Tokushi; Tolstikova, Alexandra; Xu, Weijun; Ourmazd, Abbas; Spence, John C. H.; Schwander, Peter; Barty, Anton; Chapman, Henry N.; Fromme, Petra; Mancuso, Adrian P.; Phillips, George N.; Bean, Richard; Pollack, Lois; Schmidt, Marius | Palabras clave: | European X-ray Free-Electron Laser X-ray crystallography Antibiotic resistance Ceftriaxone Drug discovery Enzyme kinetics Enzyme mechanisms Irreversible inhibition; Megahertz pulse-repetition rate Mix-and-inject serial crystallography Protein structure determination Serial femtosecond crystallography Substrate diffusion in crystals Sulbactam β-lactamases |
Fecha de publicación: | 1-nov-2021 | Editor: | International Union of Crystallography | Citación: | IUCrJ 8: 878–895 (2021) | Resumen: | Here, we illustrate what happens inside the catalytic cleft of an enzyme when substrate or ligand binds on single-millisecond timescales. The initial phase of the enzymatic cycle is observed with near-atomic resolution using the most advanced X-ray source currently available: the European XFEL (EuXFEL). The high repetition rate of the EuXFEL combined with our mix-and-inject technology enables the initial phase of ceftriaxone binding to the Mycobacterium tuberculosis β-lactamase to be followed using time-resolved crystallography in real time. It is shown how a diffusion coefficient in enzyme crystals can be derived directly from the X-ray data, enabling the determination of ligand and enzyme-ligand concentrations at any position in the crystal volume as a function of time. In addition, the structure of the irreversible inhibitor sulbactam bound to the enzyme at a 66 ms time delay after mixing is described. This demonstrates that the EuXFEL can be used as an important tool for biomedically relevant research. | Descripción: | 18 pags, 11 figs, 5 tabs | Versión del editor: | https://doi.org/10.1107/S2052252521008125 | URI: | http://hdl.handle.net/10261/258729 | DOI: | 10.1107/S2052252521008125 | ISSN: | 2052-2525 |
Aparece en las colecciones: | (IQF) Artículos |
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enzyme_crystals.pdf | artículo principal | 2,29 MB | Adobe PDF | Visualizar/Abrir |
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