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

Serial femtosecond crystallography has opened new opportunities in structural biology. Light-triggered systems have enabled time-resolved experiments, but most enzymes are not light-dependent, and many reactions rely on ligand diffusion into active sites. This work presents a drop-on-drop sample delivery system for enzyme-catalyzed reactions in microcrystal slurries. Ligand solutions are delivered as bursts of picoliter drops onto larger crystal-containing drops, inducing turbulent mixing and transporting the mixture to the X-ray interaction region with temporal control. Mixing is demonstrated using fluorescent dyes, numerical simulations, and time-resolved serial femtosecond crystallography, showing rapid ligand diffusion through microdroplets. The drop-on-drop method is widely applicable to serial crystallography, particularly for enzyme reactions with small molecule substrates.

Agata Butryn, Philipp S. Simon, Pierre Aller, Philip Hinchliffe, Ramzi N. Massad, Gabriel Leen, Catherine L. Tooke, Isabel Bogacz, In-Sik Kim, Asmit Bhowmick, Aaron S. Brewster, Nicholas E. Devenish, Jürgen Brem, Jos J. A. G. Kamps, Pauline A. Lang, Patrick Rabe, Danny Axford, John H. Beale, Bradley Davy, Ali Ebrahim, Julien Orlans, Selina L. S. Storm, Tiankun Zhou, Shigeki Owada, Rie Tanaka, Kensuke Tono, Gwyndaf Evans, Robin L. Owen, Frances A. Houle, Nicholas K. Sauter, Christopher J. Schofield, James Spencer, Vittal K. Yachandra, Junko Yano, Jan F. Kern, Allen M. Orville