skip to main content
US FlagAn official website of the United States government
dot gov icon
Official websites use .gov
A .gov website belongs to an official government organization in the United States.
https lock icon
Secure .gov websites use HTTPS
A lock ( lock ) or https:// means you've safely connected to the .gov website. Share sensitive information only on official, secure websites.
Attention:The NSF Public Access Repository (NSF-PAR) system and access will be unavailable from 7:00 AM ET to 7:30 AM ET on Friday, April 24 due to maintenance. We apologize for the inconvenience.

Explore Research Products in the PAR
It may take a few hours for recently added research products to appear in PAR search results.


  • NSF Public Access
  • Search Results
  • Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography
Title: Observation of substrate diffusion and ligand binding in enzyme crystals using high-repetition-rate mix-and-inject serial crystallography
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 theMycobacterium tuberculosisβ-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.  more » « less
Award ID(s):
1231306
PAR ID:
10587692
Author(s) / Creator(s):
; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; more » ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; ; « less
Publisher / Repository:
IUCrJ
Date Published:
Journal Name:
IUCrJ
Volume:
8
Issue:
6
ISSN:
2052-2525
Page Range / eLocation ID:
878 to 895
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. ; ; ; ; ; ; ; ; ; ; et al (, Acta Crystallographica Section F Structural Biology Communications)
    Asojo, O (Ed.)
    Burkholderia cenocepaciais an opportunistic human pathogen that can cause lethal infections in immunocompromised individuals, particularly those with cystic fibrosis. As such, there is a critical need to identify and characterize the structure and function of enzymes that participate in the metabolic pathways of this bacterium. Here, the high-resolution X-ray crystal structure of a short-chain dehydrogenase reductase (SDR) fromB. cenocepaciaJ2315 (BcSDR) in complex with the coenzyme NADP+and a benzoic acid ligand is presented. This protein has the conserved Rossmann fold of the SDR superfamily and the characteristic TGxxxGxG motif of the classical SDR subfamily. However, unlike classical SDRs, the active site of BcSDR has a leucine residue in place of the highly conserved and catalytically important tyrosine residue. Sequence analysis confirms that this leucine residue is conserved in this SDR across the Burkholderiales order. This suggests that BcSDR is more appropriately classified into the divergent SDR subfamily. In addition, this enzyme would necessarily employ a different enzyme mechanism to that proposed as a general mechanism for most SDRs. 
    more » « less
  2. (, Crystals)
    Ever since the first structure of an enzyme, lysozyme, was solved, scientists have been eager to explore how these molecules perform their catalytic function. There has been an overwhelmingly large body of publications that report the X-ray structures of enzymes determined after substrate and ligand binding. None of them truly show the structures of an enzyme working freely through a sequence of events that range from the formation of the enzyme–substrate complex to the dissociation of the product. The technical difficulties were too severe. By 1969, Sluyterman and de Graaf had pointed out that there might be a way to start a reaction in an enzyme crystal by diffusion and following its catalytic cycle in its entirety with crystallographic methods. The crystal only has to be thin enough so that the diffusion is not rate limiting. Of course, the key questions are as follows: How thin should the crystal be? Will the existing X-ray sources be able to collect data from a thin enough crystal fast enough? This review shines light on these questions. 
    more » « less
  3. ; ; ; ; ; ; ; ; ; ; et al (, The Astrophysical Journal)
    Abstract We present a detailed compilation and analysis of the X-ray phase space of low- to intermediate-redshift (0 ≤z≤ 1) transients that consolidates observed light curves (and theory where necessary) for a large variety of classes of transient/variable phenomena in the 0.3–10 keV energy band. We include gamma-ray burst afterglows, supernovae, supernova shock breakouts and shocks interacting with the environment, tidal disruption events and active galactic nuclei, fast blue optical transients, cataclysmic variables, magnetar flares/outbursts and fast radio bursts, cool stellar flares, X-ray binary outbursts, and ultraluminous X-ray sources. Our overarching goal is to offer a comprehensive resource for the examination of these ephemeral events, extending the X-ray duration–luminosity phase space (DLPS) to show luminosity evolution. We use existing observations (both targeted and serendipitous) to characterize the behavior of various transient/variable populations. Contextualizing transient signals in the larger DLPS serves two primary purposes: to identify areas of interest (i.e., regions in the parameter space where one would expect detections, but in which observations have historically been lacking), and to provide initial qualitative guidance in classifying newly discovered transient signals. We find that while the most luminous (largely extragalactic) and least luminous (largely Galactic) part of the phase space is well populated att> 0.1 days, intermediate-luminosity phenomena (LX= 1034–1042erg s−1) represent a gap in the phase space. We thus identifyLX= 1034–1042erg s−1andt= 10−4to 0.1 days as a key discovery phase space in transient X-ray astronomy. 
    more » « less
  4. ; ; ; ; ; ; (, IUCrJ)
    None (Ed.)
    Time-resolved X-ray crystallography has great promise to illuminate structure–function relations and key steps of enzymatic reactions with atomic resolution. The dominant methods for chemically-initiated reactions require complex instrumentation at the X-ray beamline, significant effort to operate and maintain this instrumentation, and enormous numbers (∼105–109) of crystals per time point. We describe instrumentation and methods that enable high-throughput time-resolved study of biomolecular systems using standard crystallography sample supports and mail-in X-ray data collection at standard high-throughput cryocrystallography synchrotron beamlines. The instrumentation allows rapid reaction initiation by mixing of crystals and substrate/ligand solution, rapid capture of structural states via thermal quenching with no pre-cooling perturbations, and yields time resolutions in the single-millisecond range, comparable to the best achieved by any non-photo-initiated method in both crystallography and cryo-electron microscopy. Our approach to reaction initiation has the advantages of simplicity, robustness, low cost, adaptability to diverse ligand solutions and small minimum volume requirements, making it well suited to routine laboratory use and to high-throughput screening. We report the detailed characterization of instrument performance, present structures of binding ofN-acetylglucosamine to lysozyme at time points from 8 ms to 2 s determined using only one crystal per time point, and discuss additional improvements that will push time resolution toward 1 ms. 
    more » « less
  5. ; ; ; ; ; ; ; ; ; ; et al (, IUCrJ)
    The upgrade of the European Synchrotron Radiation Facility (ESRF) in Grenoble, France to an Extremely Brilliant Source (EBS) is expected to enable time-resolved synchrotron serial crystallography (SSX) experiments with sub-millisecond time resolution. ID29 is a new beamline dedicated to SSX experiments at ESRF–EBS. Here, we report experiments emerging from the initial phase of user operation at ID29. We first used microcrystals of photoactive yellow protein as a model system to exploit the potential of microsecond pulses for SSX. Subsequently, we investigated microcrystals of cytochromecnitrite reductase (ccNiR) with microsecond X-ray pulses. CcNiR is a decaheme protein that is ideal for the investigation of radiation damage at the various heme-iron sites. Finally, we performed a proof-of-concept subsecond time-resolved SSX experiment by photoactivating microcrystals of a myxobacterial phytochrome. 
    more » « less
  • Citation Formats
  • MLA
  • APA
  • Chicago
  • BibTeX
  • Text Encoded Conversion
  • XML
  • Save / Share this Record
  • Send to Email