Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation.
Serial millisecond crystallography of membrane and soluble protein microcrystals using synchrotron radiation.
复制标题
使用同步加速器辐射的膜和可溶性蛋白微晶的连续毫秒晶体学。
DOI：
10.1107/s205225251700570x
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发表时间：
2017-07-01
期刊：
影响因子：
3.9
通讯作者：
Liu W
中科院分区：
文献类型：
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作者：
Martin-Garcia JM;Conrad CE;Nelson G;Stander N;Zatsepin NA;Zook J;Zhu L;Geiger J;Chun E;Kissick D;Hilgart MC;Ogata C;Ishchenko A;Nagaratnam N;Roy-Chowdhury S;Coe J;Subramanian G;Schaffer A;James D;Ketwala G;Venugopalan N;Xu S;Corcoran S;Ferguson D;Weierstall U;Spence JCH;Cherezov V;Fromme P;Fischetti RF;Liu W
在这项原理验证研究中，使用连续毫秒晶体学（SMX）的几种蛋白质的结构测定的可行性进行了评估。报道了在美国同步辐射光源先进光子源（APS）上进行的第一个基于高粘度注入器的SMX实验。由于X射线自由电子激光（XFEL）光源的缺乏，利用连续飞秒晶体学（SFX）新技术测定生物大分子的晶体结构受到严重限制。然而，最近和未来的升级使微焦点光束线在同步辐射源适合室温连续晶体学数据收集也。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长，到目前为止已经报道了十几个实验。在这里，第一个高粘度注射器为基础的SMX实验进行了美国同步加速器源，先进的光子源（APS），报告。筛选了多种蛋白质的微晶（5-20µm），包括溶菌酶、索马甜、藻蓝蛋白、人A2 A腺苷受体（A2 AAR）、膜脂蛋白Flpp 3的可溶性片段和蛋白酶K。晶体悬浮在无规立方相（LCP）或高分子量聚（环氧乙烷）（PEO;分子量8 000 000）被交付到使用高粘度注射器的光束。 APS开发的内部数据缩减（命中发现）软件以及SFX数据缩减和分析软件套件Cheetah和CrystFEL实现了有效的现场SMX数据监测、缩减和处理。收集了A2 AAR、藻蓝蛋白、Flpp 3、蛋白酶K和溶菌酶的完整数据集，并分别在3.2、3.1、2.65和2.05 μ m分辨率下测定了A2 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使用高粘度注射器从5-20 µm晶体进行连续毫秒晶体学的可行性。 本研究中获得的晶体结构的分辨率取决于电流通量密度和晶体尺寸，但光束线光学的即将发展和计划中的APS-U升级将使强度增加两个数量级。这些发展将使结构测定从较小的和/或弱衍射微晶。
In this proof-of-principle study, the feasibility of structure determination of several proteins using serial millisecond crystallography (SMX) has been evaluated. The first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. 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.