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

DOI: 10.1038/s41467-021-24757-7

Authors: Agata Butryn (Diamond Light Source; Research Complex at Harwell) , Philipp S. Simon (Lawrence Berkeley National Laboratory) , Pierre Aller (Diamond Light Source; Research Complex at Harwell) , Philip Hinchliffe (University of Bristol) , Ramzi N. Massad (Lawrence Berkeley National Laboratory) , Gabriel Leen (PolyPico Technologies Ltd; University of Limerick) , Catherine L. Tooke (University of Bristol) , Isabel Bogacz (Lawrence Berkeley National Laboratory) , In-Sik Kim (Lawrence Berkeley National Laboratory) , Asmit Bhowmick (Lawrence Berkeley National Laboratory) , Aaron S. Brewster (Lawrence Berkeley National Laboratory) , Nicholas E. Devenish (Diamond Light Source) , Jurgen Brem (University of Oxford) , Jos J. A. G. Kamps (Diamond Light Source; University of Oxford) , Pauline A. Lang (University of Oxford) , Patrick Rabe (University of Oxford) , Danny Axford (Diamond Light Source) , John H. Beale (Diamond Light Source) , Bradley Davy (Diamond Light Source) , Ali Ebrahim (Diamond Light Source) , Julien Orlans (Diamond Light Source; University of Lyon) , Selina L. S. Storm (Diamond Light Source) , Tiankun Zhou (Diamond Light Source; Research Complex at Harwell) , Shigeki Owada (RIKEN SPring-8 Center; Japan Synchrotron Radiation Research Institute) , Rie Tanaka (RIKEN SPring-8 Center; Kyoto University) , Kensuke Tono (RIKEN SPring-8 Center; Japan Synchrotron Radiation Research Institute) , Gwyndaf Evans (Diamond Light Source) , Robin L. Owen (Diamond Light Source) , Frances A. Houle (Lawrence Berkeley National Laboratory) , Nicholas K. Sauter (Lawrence Berkeley National Laboratory) , Christopher J. Schofield (University of Oxford) , James Spencer (University of Bristol) , Vittal K. Yachandra (Lawrence Berkeley National Laboratory) , Junko Yano (Lawrence Berkeley National Laboratory) , Jan F. Kern (Lawrence Berkeley National Laboratory) , Allen M. Orville (Diamond Light Source; Research Complex at Harwell)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Nature Communications , VOL 12

State: Published (Approved)
Published: July 2021
Diamond Proposal Number(s): 19458 , 25260

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.

Journal Keywords: Computational models Optical spectroscopy; Structural biology

Diamond Keywords: Enzymes

Subject Areas: Biology and Bio-materials, Chemistry, Technique Development

Diamond Offline Facilities: XFEL-Hub
Instruments: I24-Microfocus Macromolecular Crystallography

Other Facilities: BL2 at SACLA

Added On: 26/07/2021 13:30

Documents:
s41467-021-24757-7.pdf

Discipline Tags:

Biochemistry Catalysis Technique Development - Life Sciences & Biotech Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)