2. Academic Validation
  3. An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

An on-demand, drop-on-drop method for studying enzyme catalysis by serial crystallography

  • Nat Commun. 2021 Jul 22;12(1):4461. doi: 10.1038/s41467-021-24757-7.
Agata Butryn  # 1 2,  Philipp S Simon  # 3,  Pierre Aller  # 1 2,  Philip Hinchliffe 4,  Ramzi N Massad 3,  Gabriel Leen 5 6,  Catherine L Tooke 4,  Isabel Bogacz 3,  In-Sik Kim 3,  Asmit Bhowmick 3,  Aaron S Brewster 3,  Nicholas E Devenish 1,  Jürgen Brem 7,  Jos J A G Kamps 7 1,  Pauline A Lang 7,  Patrick Rabe 7,  Danny Axford 1,  John H Beale 1 8,  Bradley Davy 1 9,  Ali Ebrahim 1,  Julien Orlans 1 10,  Selina L S Storm 1 11,  Tiankun Zhou 1 2,  Shigeki Owada 12 13,  Rie Tanaka 12 14,  Kensuke Tono 12 13,  Gwyndaf Evans 1,  Robin L Owen 1,  Frances A Houle 15,  Nicholas K Sauter 3,  Christopher J Schofield 7,  James Spencer 4,  Vittal K Yachandra 3,  Junko Yano 3,  Jan F Kern 16,  Allen M Orville 17 18
Affiliations

Affiliations

  • 1 Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK.
  • 2 Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK.
  • 3 Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
  • 4 School of Cellular and Molecular Medicine, University of Bristol, University Walk, Bristol, UK.
  • 5 PolyPico Technologies Ltd, Unit 10, Airways Technology Park, Rathmacullig West, Cork, Ireland.
  • 6 Department of Electronic and Computer Engineering, University of Limerick, Limerick, Ireland.
  • 7 Department of Chemistry, Chemistry Research Laboratory, University of Oxford, Oxford, UK.
  • 8 Paul Scherrer Institut, Villigen PSI, Switzerland.
  • 9 School of Computing, University of Leeds, Leeds, UK.
  • 10 UMR0203, Biologie Fonctionnelle, Insectes et Interactions, Institut National des Sciences Appliquées de Lyon, Institut National de Recherche pour l'Agriculture, l'Alimentation et l'Environnement, University of Lyon, Villeurbanne, France.
  • 11 European Molecular Biology Laboratory, Hamburg Outstation c/o DESY, Hamburg, Germany.
  • 12 RIKEN SPring-8 Center, Hyogo, Japan.
  • 13 Japan Synchrotron Radiation Research Institute, Hyogo, Japan.
  • 14 Department of Cell Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
  • 15 Chemical Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA.
  • 16 Molecular Biophysics and Integrated Bioimaging Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA. jfkern@lbl.gov.
  • 17 Diamond Light Source, Harwell Science and Innovation Campus, Didcot, UK. allen.orville@diamond.ac.uk.
  • 18 Research Complex at Harwell, Rutherford Appleton Laboratory, Didcot, UK. allen.orville@diamond.ac.uk.
  • # Contributed equally.
PMID: 34294694 DOI: 10.1038/s41467-021-24757-7
Abstract

Serial femtosecond crystallography has opened up many new opportunities in structural biology. In recent years, several approaches employing light-inducible systems have emerged to enable time-resolved experiments that reveal protein dynamics at high atomic and temporal resolutions. However, very few Enzymes are light-dependent, whereas macromolecules requiring ligand diffusion into an active site are ubiquitous. In this work we present a drop-on-drop sample delivery system that enables the study of enzyme-catalyzed reactions in microcrystal slurries. The system delivers ligand solutions in bursts of multiple picoliter-sized drops on top of a larger crystal-containing drop inducing turbulent mixing and transports the mixture to the X-ray interaction region with temporal resolution. We demonstrate mixing using fluorescent dyes, numerical simulations and time-resolved serial femtosecond crystallography, which show rapid ligand diffusion through microdroplets. The drop-on-drop method has the potential to be widely applicable to serial crystallography studies, particularly of Enzyme reactions with small molecule substrates.

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