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Jose M. Martin-Garcia and Chelsie E. Conrad and Garrett Nelson and Natasha Stander and Nadia A. Zatsepin and James Zook and Lan Zhu and James Geiger and Eugene Chun and David Kissick and Mark C. Hilgart and Craig Ogata and Andrii Ishchenko and Nirupa Nagaratnam and Shatabdi Roy-Chowdhury and Jesse Coe and Ganesh Subramanian and Alexander Schaffer and Daniel James and Gihan Ketwala and Nagarajan Venugopalan and Shenglan Xu and Stephen Corcoran and Dale Ferguson and Uwe Weierstall and John C. H. Spence and Vadim Cherezov and Petra Fromme and Robert F. Fischetti and Wei Liu

Martin-Garcia, J.M. et al., 2017. Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation. IUCrJ, 4(4). Available at: http://dx.doi.org/10.1107/s205225251700570x.

Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A2A adenosine receptor (A2AAR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A2AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A2AAR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.

http://dx.doi.org/10.1107/s205225251700570x

NSF-STC Biology with X-ray Lasers (NSF-1231306)