Martin-Garcia Jose M, Conrad Chelsie E, Nelson Garrett, Stander Natasha, Zatsepin Nadia A, Zook James, Zhu Lan, Geiger James, Chun Eugene, Kissick David, Hilgart Mark C, Ogata Craig, Ishchenko Andrii, Nagaratnam Nirupa, Roy-Chowdhury Shatabdi, Coe Jesse, Subramanian Ganesh, Schaffer Alexander, James Daniel, Ketwala Gihan, Venugopalan Nagarajan, Xu Shenglan, Corcoran Stephen, Ferguson Dale, Weierstall Uwe, Spence John C H, Cherezov Vadim, Fromme Petra, Fischetti Robert F, Liu Wei
School of Molecular Sciences and Biodesign Center for Applied Structural Discovery, Biodesign Institute, Arizona State University, Tempe, AZ 85287, USA.
Structural Biophysics Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
IUCrJ. 2017 May 24;4(Pt 4):439-454. doi: 10.1107/S205225251700570X. eCollection 2017 Jul 1.
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 A adenosine receptor (AAR), 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 and enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for AAR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of AAR, 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.
使用串行飞秒晶体学（SFX）这项新技术来确定生物大分子的晶体结构，受到X射线自由电子激光（XFEL）源稀缺的严重限制。然而，近期及未来的升级使得同步辐射源处的微聚焦光束线也适用于室温串行晶体学数据收集。由于同步加速器需要更长的曝光时间，串行数据收集被称为串行毫秒晶体学（SMX）。因此，SMX实验的数量正在迅速增加，到目前为止已有十几个实验被报道。在此，报道了在美国同步辐射源——先进光子源（APS）上进行的首次基于高粘度注射器的SMX实验。对多种蛋白质的微晶（5 - 20 µm）进行了筛选，这些蛋白质包括溶菌酶、奇异果甜蛋白、藻蓝蛋白、人A腺苷受体（AAR）、膜脂蛋白Flpp3的可溶性片段和蛋白酶K。悬浮在脂质立方相（LCP）或高分子量聚环氧乙烷（PEO；分子量8 000 000）中的晶体，使用高粘度注射器输送到光束处。APS开发的内部数据处理（寻峰）软件以及SFX数据处理和分析软件套件，实现了高效的现场SMX数据监测、处理和分析。为AAR、藻蓝蛋白、Flpp3、蛋白酶K和溶菌酶收集了完整的数据集，并且分别以3.2 Å、3.1 Å、2.65 Å和2.05 Å的分辨率确定了AAR、藻蓝蛋白、蛋白酶K和溶菌酶的结构。这些数据证明了在APS使用高粘度注射器对5 - 20 µm晶体进行串行毫秒晶体学的可行性。本研究中获得的晶体结构分辨率受当前通量密度和晶体尺寸的限制，但光束线光学的未来发展以及计划中的APS - U升级将使强度提高两个数量级。这些发展将能够从小的和/或弱衍射微晶中确定结构。
