X-ray free-electron lasers (XFELs) provide very intense X-ray pulses suitable for macromolecular crystallography. Each X-ray pulse typically lasts for tens of femtoseconds and the interval between pulses is many orders of magnitude longer. Here we describe two novel acoustic injection systems that use focused sound waves to eject picoliter to nanoliter crystal-containing droplets out of microplates and into the X-ray pulse from which diffraction data are collected. The on-demand droplet delivery is synchronized to the XFEL pulse scheme, resulting in X-ray pulses intersecting up to 88% of the droplets. We tested several types of samples in a range of crystallization conditions, wherein the overall crystal hit ratio (e.g., fraction of images with observable diffraction patterns) is a function of the microcrystal slurry concentration. We report crystal structures from lysozyme, thermolysin, and stachydrine demethylase (Stc2). Additional samples were screened to demonstrate that these methods can be applied to rare samples.
Bibliographical noteFunding Information:
Experiments were carried out at the LCLS, a national user facility operated by Stanford University on behalf of the US Department of Energy (DOE), Office of Basic Energy Sciences (OBES). Use of the LCLS, SLAC National Accelerator Laboratory, is supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. DE-AC02-76SF00515 . We thank the staff at LCLS/SLAC for their support. This work was supported by the Brookhaven National Laboratory/US DOE , Laboratory Directed Research and Development grant 11-008 (A.S.S., M.A., and A.M.O.); NIH/NCRR grant 2-P41-RR012408 , NIH/National Institute of General Medical Sciences (NIGMS) grant 8P41GM103473-16 and the US DOE, Office of Biological and Environmental Research (OBER) grant FWP BO-70 (A.H., A.M.O., and A.S.S); NIH NIGMS grant Y1GM008003 (M.A.); and the Brookhaven National Laboratory Biosciences Department grant BO-9734 (R.A.). NIH grants GM095887 and GM102520 and Director, Office of Science, DOE under contract DE-AC02-05CH11231 for data-processing methods (A.S.B. and N.K.S.). NIH 5R01 GM066569-11 (C.M.W.), NIH 5R37 GM041574-26 (V.L.D.), NIH F32 GM097779-03 (E.T.Y.) for MauG-MADH biochemistry. Anaerobically purified Stc2 production was supported by the National Science Foundation grant CHE 0748504 (P.L.). PS-II production and crystallization was supported by the US DOE Director, Office of Science, OBES, Division of Chemical Sciences, Geosciences, and Biosciences (CSGB) under contract DE-AC02-05CH11231 (J.Y. and V.K.Y.); NIH grants GM055302 (V.K.Y.) and GM110501 (J.Y.), the Human Frontiers Science Project (J.Y. and A.Z.), the DFG-Cluster of Excellence “UniCat” coordinated by the Technische Universität Berlin and Sfb1078, TP A5 (A.Z.). Testing of crystals and various parts of the setup were carried out at synchrotron facilities that were provided by the Advanced Light Source (ALS) in Berkeley, the National Synchrotron Light Source (NSLS) in Upton, NY, and the Stanford Synchrotron Radiation Lightsource (SSRL). Use of the National Synchrotron Light Source, Brookhaven National Laboratory, was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences , under contract no. DE-AC02-98CH10886 . Use of the Stanford Synchrotron Radiation Lightsource, SLAC National Accelerator Laboratory, is supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under contract no. DE-AC02-76SF00515 . The SSRL Structural Molecular Biology Program is supported by the DOE Office of Biological and Environmental Research , and by the NIH, National Institute of General Medical Sciences (including P41GM103393 ). Richard Ellson is a founder and a member of the Board of Directors of Labcyte Inc. S.D., B.E., L.P.G., L.M., W.M.M., J.O., and R.G.S. are employed by Labcyte Inc.