Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography

Suraj Pandey, George Calvey, Andrea M. Katz, Tek Narsingh Malla, Faisal H.M. Koua, Jose M. Martin-Garcia, Ishwor Poudyal, Jay How Yang, Mohammad Vakili, Oleksandr Yefanov, Kara A. Zielinski, Sasa Bajt, Salah Awel, Katarina Doerner, Matthias Frank, Luca Gelisio, Rebecca Jernigan, Henry Kirkwood, Marco Kloos, Jayanath KoliyaduValerio Mariani, Mitchell D. Miller, Grant Mills, Garrett Nelson, Jose L. Olmos, Alireza Sadri, Tokushi Sato, Alexandra Tolstikova, Weijun Xu, Abbas Ourmazd, John C.H. Spence, Peter Schwander, Anton Barty, Henry N. Chapman, Petra Fromme, Adrian P. Mancuso, George N. Phillips, Richard Bean, Lois Pollack, Marius Schmidt

Research output: Contribution to journalArticlepeer-review

33 Scopus citations


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.

Original languageEnglish (US)
Pages (from-to)878-895
Number of pages18
StatePublished - Nov 1 2021


  • Antibiotic resistance
  • Ceftriaxone
  • Drug discovery
  • Enzyme kinetics
  • Enzyme mechanisms
  • European X-ray Free-Electron Laser
  • Irreversible inhibition
  • Megahertz pulse-repetition rate
  • Mix-and-inject serial crystallography
  • Protein structure determination
  • Serial femtosecond crystallography
  • Substrate diffusion in crystals
  • Sulbactam
  • X-ray crystallography
  • β-lactamases

ASJC Scopus subject areas

  • Chemistry(all)
  • Biochemistry
  • Materials Science(all)
  • Condensed Matter Physics


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