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

Pandey, 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, Jr, 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, Jr, George N.; Bean, Richard; Pollack, Lois; Schmidt, Marius

doi:10.1107/S2052252521008125

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

Full Record
Other Related Research

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.

Authors:

Pandey, Suraj; Calvey, George; Katz, Andrea M.; Malla, Tek Narsingh;

; Martin-Garcia, Jose M.; Poudyal, Ishwor; Yang, Jay-How;

;

; Doerner, Katarina; Frank, Matthias; Gelisio, Luca; Jernigan, Rebecca; Kirkwood, Henry; Kloos, Marco; Koliyadu, Jayanath more »

Publication Date:: Thu Sep 09 00:00:00 EDT 2021

Research Org.:: Lawrence Livermore National Laboratory (LLNL), Livermore, CA (United States); SLAC National Accelerator Laboratory (SLAC), Menlo Park, CA (United States)

Sponsoring Org.:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Science Foundation (NSF); National Institutes of Health (NIH); German Research foundation (DFG); European Union 2020; European Research Council (ERC); USDOE National Nuclear Security Administration (NNSA)

OSTI Identifier:: 1819234

Alternate Identifier(s):: OSTI ID: 1904218; OSTI ID: 2204475

Report Number(s):: LLNL-JRNL-856757
Journal ID: ISSN 2052-2525; IUCRAJ; PII: S2052252521008125

Grant/Contract Number:: AC52-07NA27344; SC0002164; STC-1231306; 1551489; DBI-2029533; 1450681; R01 GM117342-0404; R01 GM095583; 1565180; T32-GM008267; 637295; ERC-2013-SyG 609920; RGP0010 2017; 609920

Resource Type:: Published Article

Journal Name:: IUCrJ

Additional Journal Information:: Journal Name: IUCrJ Journal Volume: 8 Journal Issue: 6; Journal ID: ISSN 2052-2525

Publisher:: International Union of Crystallography (IUCr)

Country of Publication:: United Kingdom

Language:: English

Subject:: 36 MATERIALS SCIENCE; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; substrate diffusion in crystals; antibiotic resistance; β-lactamases; enzyme kinetics; irreversible inhibition; mix-and-inject serial crystallography; serial femtosecond crystallography; European X-ray Free-Electron Laser; megahertz pulse-repetition rate; protein structure determination; drug discovery; ceftriaxone; sulbactam; X-ray crystallography; enzyme mechanisms

Citation Formats


                    Pandey, 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, Jr, 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, Jr, George N., Bean, Richard, Pollack, Lois, and Schmidt, Marius. Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography.  United Kingdom: N. p., 2021. 
Web.  doi:10.1107/S2052252521008125.

Copy to clipboard


                    Pandey, 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, Jr, 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, Jr, George N., Bean, Richard, Pollack, Lois, & Schmidt, Marius. Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography.  United Kingdom.  https://doi.org/10.1107/S2052252521008125

Copy to clipboard


                    Pandey, 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, Jr, 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, Jr, George N., Bean, Richard, Pollack, Lois, and Schmidt, Marius. Thu .  
"Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography".  United Kingdom.  https://doi.org/10.1107/S2052252521008125.

Copy to clipboard


                    
@article{osti_1819234,

  title        = {Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography},

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

  abstractNote = {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          = {10.1107/S2052252521008125},

  journal      = {IUCrJ},

  number       = 6,

  volume       = 8,

  place        = {United Kingdom},

  year         = {Thu Sep 09 00:00:00 EDT 2021},

  month        = {Thu Sep 09 00:00:00 EDT 2021}

}

Copy to clipboard

Journal Article:

Free Publicly Available Full Text

Publisher's Version of Record
https://doi.org/10.1107/S2052252521008125

Other availability

Search WorldCat to find libraries that may hold this journal

Save / Share:

Export Metadata

Save to My Library

Similar Records in DOE PAGES and OSTI.GOV collections:

Time-resolved serial femtosecond crystallography at the European XFEL

Journal Article Pandey, Suraj ; Bean, Richard ; Sato, Tokushi ; ... - Nature Methods

The European XFEL (EuXFEL) is a 3.4-km long X-ray source, which produces femtosecond, ultrabrilliant and spatially coherent X-ray pulses at megahertz (MHz) repetition rates. This X-ray source has been designed to enable the observation of ultrafast processes with near-atomic spatial resolution. Time-resolved crystallographic investigations on biological macromolecules belong to an important class of experiments that explore fundamental and functional structural displacements in these molecules. Due to the unusual MHz X-ray pulse structure at the EuXFEL, these experiments are challenging. In this paper, we demonstrate how a biological reaction can be followed on ultrafast timescales at the EuXFEL. We investigate themore »« less
Cited by 82
https://doi.org/10.1038/s41592-019-0628-z

Full Text Available
Heterogeneity in M. tuberculosis β-lactamase inhibition by Sulbactam

Journal Article Malla, Tek Narsingh ; Zielinski, Kara ; Aldama, Luis ; ... - Nature Communications

Abstract For decades, researchers have elucidated essential enzymatic functions on the atomic length scale by tracing atomic positions in real-time. Our work builds on possibilities unleashed by mix-and-inject serial crystallography (MISC) at X-ray free electron laser facilities. In this approach, enzymatic reactions are triggered by mixing substrate or ligand solutions with enzyme microcrystals. Here, we report in atomic detail (between 2.2 and 2.7 Å resolution) by room-temperature, time-resolved crystallography with millisecond time-resolution (with timepoints between 3 ms and 700 ms) how the Mycobacterium tuberculosis enzyme BlaC is inhibited by sulbactam (SUB). Our results reveal ligand binding heterogeneity, ligand gating, cooperativity, induced fit, andmore »« less
https://doi.org/10.1038/s41467-023-41246-1
Crystal structure of CO-bound cytochrome c oxidase determined by serial femtosecond X-ray crystallography at room temperature

Journal Article Ishigami, Izumi ; Zatsepin, Nadia A. ; Hikita, Masahide ; ... - Proceedings of the National Academy of Sciences of the United States of America

Here, cytochrome c oxidase (CcO), the terminal enzyme in the electron transfer chain, translocates protons across the inner mitochondrial membrane by harnessing the free energy generated by the reduction of oxygen to water. Several redox-coupled proton translocation mechanisms have been proposed, but they lack confirmation, in part from the absence of reliable structural information due to radiation damage artifacts caused by the intense synchrotron radiation. Here we report the room temperature, neutral pH (6.8), damage-free structure of bovine CcO (bCcO) in the carbon monoxide (CO)-bound state at a resolution of 2.3 Å, obtained by serial femtosecond X-ray crystallography (SFX) withmore »« less
Cited by 36
https://doi.org/10.1073/pnas.1705628114

Full Text Available
Retrieving functional pathways of biomolecules from single-particle snapshots

Journal Article Dashti, Ali ; Mashayekhi, Ghoncheh ; Shekhar, Mrinal ; ... - Nature Communications

Abstract A primary reason for the intense interest in structural biology is the fact that knowledge of structure can elucidate macromolecular functions in living organisms. Sustained effort has resulted in an impressive arsenal of tools for determining the static structures. But under physiological conditions, macromolecules undergo continuous conformational changes, a subset of which are functionally important. Techniques for capturing the continuous conformational changes underlying function are essential for further progress. Here, we present chemically-detailed conformational movies of biological function, extracted data-analytically from experimental single-particle cryo-electron microscopy (cryo-EM) snapshots of ryanodine receptor type 1 (RyR1), a calcium-activated calcium channel engaged inmore »« less
https://doi.org/10.1038/s41467-020-18403-x
Enzyme intermediates captured “on the fly” by mix-and-inject serial crystallography

Journal Article Olmos, Jr, Jose L. ; Pandey, Suraj ; Martin-Garcia, Jose M. ; ... - BMC Biology

Background: Ever since the first atomic structure of an enzyme was solved, the discovery of the mechanism and dynamics of reactions catalyzed by biomolecules has been the key goal for the understanding of the molecular processes that drive life on earth. Despite a large number of successful methods for trapping reaction intermediates, the direct observation of an ongoing reaction has been possible only in rare and exceptional cases. Results: Here, we demonstrate a general method for capturing enzyme catalysis “in action” by mix-and-inject serial crystallography (MISC). Specifically, we follow the catalytic reaction of the Mycobacterium tuberculosis β-lactamase with the third-generationmore »« less
Cited by 80
https://doi.org/10.1186/s12915-018-0524-5

Similar Records