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.
Bibliographical noteFunding Information:
The authors acknowledge the financial support of this work from the National Institutes of Health (NIH) grants GM117126 (to N.K.S.), GM55302 (to V.K.Y.), GM110501 (to J. Y.), GM126289 (to J.K.), and NIH training grant GM133081 (to K.D.S.). Support was provided by the Biotechnology and Biological Sciences Research Council Grant 102593 (to A.M.O.); Wellcome Investigator Award in Science 210734/Z/18/Z (to A.M.O.); Royal Society Wolfson Fellowship RSWF\R2\182017 (to A.M.O.) and by the Director, Office of Science, Office of Basic Energy Sciences (OBES), Division of Chemical Sciences, Geosciences, and Biosciences of the Department of Energy (DOE) (to J.K., J.Y., and V.K.Y.). F.A.H. was supported by the Laboratory Directed Research and Development Program of the Department of Energy’s Lawrence Berkeley National Laboratory under DOE OBES under Contract No. DE-AC02-05CH11231. C.L.T. was supported by BBSRC-funded South West Biosciences Doctoral Training Partnership (BB/J014400/1). J.J.A.G.K. was funded by the EPSRC Synthesis 345 for Biology and Medicine CDT(EP/L015838/1) and a Clarendon Scholarship. R.T. was supported by Platform Project for Supporting Drug Discovery and Life Science Research (Basis for Supporting Innovative Drug Discovery and Life Science Research (BINDS)) from AMED under Grant Number JP20am0101070. XFEL data was collected under proposal 2019A8088 at the BL2 instrument at SACLA, Japan. Fixed-target data was collected under proposal mx19458 and mx25260 at Diamond Light Source BLI24.
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