Journal ArticleObservation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallographyPandey, Suraj; Calvey, George; Katz, Andrea M; Malla, Tek Narsingh; Koua, Faisal_H M; Martin-Garcia, Jose M; 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<p>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<italic>Mycobacterium tuberculosis</italic>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.</p>IUCrJ2021-11-0110587692IUCrJ86878 to 8952052-2525https://doi.org/10.1107/s20522525210081251231306National Science Foundation