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

By Suraj Pandey1, George Douglas Calvey, Andrea Katz, Tek Narsingh Malla1, Faisal Hammad Mekky Koua2, Jose M. Martin-Garcia, Ishwor Poudyal1, Jay-How Yang3, Mohammad Vakili, Oleksandr Yefanov, Kara Zielinski4, Sasa Bajt, Salah Awel, Katarina Doerner, Matthias Frank5, Luca Gelisio, Rebecca Jeanne Jernigan3, Henry Kirkwood, Marco Kloos, Jayanath Koliyadu, Valerio Mariani, Mitchell Miller6, Grant Mills, Garrett Charles Nelson3, Jose L. Olmos, Alireza Sadri, Tokushi Sato, Alexandra Tolstikova, Weijun Xu, Abbas Ourmazd7, John Spence3, Peter Schwander7, Anton Barty, Henry Chapman4, Petra Fromme3, Adrian P. Mancuso, George Phillips6, Richard Bean, Lois Pollack8, Marius Schmidt1

1. University of Wisconsin - Milwaukee 2. Nagoya Institute of Technology 3. Arizona State University 4. Center for Free-Electron Laser Science 5. Lawrence Livermore National Laboratory 6. Rice University 7. University of Wisconsin-Milwaukee 8. Cornell University

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Type

journal-article

Author

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

Citation

Pandey, S. et al., 2021. Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography. IUCrJ, 8(6). Available at: http://dx.doi.org/10.1107/s2052252521008125.

Abstract

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.

DOI

Funding

NSF-STC Biology with X-ray Lasers (NSF-1231306)