| Literature DB >> 34294694 |
Agata Butryn1,2, Philipp S Simon3, Pierre Aller1,2, Philip Hinchliffe4, Ramzi N Massad3, Gabriel Leen5,6, Catherine L Tooke4, Isabel Bogacz3, In-Sik Kim3, Asmit Bhowmick3, Aaron S Brewster3, Nicholas E Devenish1, Jürgen Brem7, Jos J A G Kamps7,1, Pauline A Lang7, Patrick Rabe7, Danny Axford1, John H Beale1,8, Bradley Davy1,9, Ali Ebrahim1, Julien Orlans1,10, Selina L S Storm1,11, Tiankun Zhou1,2, Shigeki Owada12,13, Rie Tanaka12,14, Kensuke Tono12,13, Gwyndaf Evans1, Robin L Owen1, Frances A Houle15, Nicholas K Sauter3, Christopher J Schofield7, James Spencer4, Vittal K Yachandra3, Junko Yano3, Jan F Kern16, Allen M Orville17,18.
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.Entities:
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Year: 2021 PMID： 34294694 DOI： 10.1038/s41467-021-24757-7
Source DB: PubMed Journal: Nat Commun ISSN： 2041-1723 Impact factor: 14.919