Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal.
Serial crystallography captures enzyme catalysis in copper nitrite reductase at atomic resolution from one crystal.
复制标题
系列晶体学以原子分辨率从一个晶体捕获亚硝酸铜还原酶的酶催化作用。
DOI：
10.1107/s205225251600823x
复制
发表时间：
2016-07-01
期刊：
影响因子：
3.9
通讯作者：
Strange RW
中科院分区：
文献类型：
--
作者：
Horrell S;Antonyuk SV;Eady RR;Hasnain SS;Hough MA;Strange RW
连续晶体学已用于驱动亚硝酸铜还原酶通过其酶循环，同时对相同体积的单个低温维持晶体进行采样。由此获得了 X 射线驱动酶催化的结构影片，以前所未有的细节揭示了催化反应过程中发生的结构变化。将单个蛋白质晶体结构与酶机制联系起来仍然是结构生物学的一个主要且具有挑战性的目标。最近在同步辐射和 X 射线自由电子激光实验中报道了使用多个晶体的串行晶体学。在这项工作中，使用连续晶体学从一个晶体（MSOX）连续获得多个结构，以研究晶体酶催化。利用同步加速器 MX 光束线上的快速、无快门 X 射线检测器技术对单个亚硝酸铜还原酶晶体进行低剂量连续晶体学，该晶体的存活时间足够长，可以以 1.07–1.62 Å 的分辨率收集 45 个连续的 100 K X 射线结构，所有这些都从相同的晶体体积中采样。这种系列晶体学方法揭示了在 X 射线产生的溶剂化电子还原 1 型 Cu 电子转移中心后，结合在催化 2 型 Cu 中心的底物逐渐从亚硝酸盐转化为一氧化氮。当酶经历其催化循环（即亚硝酸盐还原）时，活性位点中明显的显着且明确的结构重排在整个反硝化过程中是至关重要的一步。建议这种连续晶体学方法广泛适用于研究单个蛋白质晶体的任何氧化还原或电子驱动的酶反应。它可以提供一个“催化反应电影”，突出显示酶催化过程中发生的结构变化。数据分析和建模自动化的预期发展可能会允许在一些强大的同步加速器晶体光束线现场对此类数据进行无缝和近实时的分析。
Serial crystallography has been used to drive copper nitrite reductase through its enzymatic cycle while sampling the same volume of a single cryogenically maintained crystal. A structural movie of X-ray-driven enzyme catalysis has thus been obtained, revealing the structural changes that occur during the catalytic reaction in unprecedented detail. Relating individual protein crystal structures to an enzyme mechanism remains a major and challenging goal for structural biology. Serial crystallography using multiple crystals has recently been reported in both synchrotron-radiation and X-ray free-electron laser experiments. In this work, serial crystallography was used to obtain multiple structures serially from one crystal (MSOX) to study in crystallo enzyme catalysis. Rapid, shutterless X-ray detector technology on a synchrotron MX beamline was exploited to perform low-dose serial crystallography on a single copper nitrite reductase crystal, which survived long enough for 45 consecutive 100 K X-ray structures to be collected at 1.07–1.62 Å resolution, all sampled from the same crystal volume. This serial crystallography approach revealed the gradual conversion of the substrate bound at the catalytic type 2 Cu centre from nitrite to nitric oxide, following reduction of the type 1 Cu electron-transfer centre by X-ray-generated solvated electrons. Significant, well defined structural rearrangements in the active site are evident in the series as the enzyme moves through its catalytic cycle, namely nitrite reduction, which is a vital step in the global denitrification process. It is proposed that such a serial crystallography approach is widely applicable for studying any redox or electron-driven enzyme reactions from a single protein crystal. It can provide a ‘catalytic reaction movie’ highlighting the structural changes that occur during enzyme catalysis. The anticipated developments in the automation of data analysis and modelling are likely to allow seamless and near-real-time analysis of such data on-site at some of the powerful synchrotron crystallographic beamlines.