Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent
Membrane protein structure determination by SAD, SIR, or SIRAS phasing in serial femtosecond crystallography using an iododetergent
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DOI：
10.1073/pnas.1602531113
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发表时间：
2016-11-15
影响因子：
11.1
通讯作者：
Mizohata, Eiichi
中科院分区：
文献类型：
--
作者：
Nakane, Takanori;Hanashima, Shinya;Mizohata, Eiichi
用x射线晶体学测定生物大分子的三维结构存在相位问题：为了进行傅里叶变换来计算实际空间密度图，结构因子的强度和相位都是必需的；然而，测量的衍射图只能给出强度。尽管自2009年以来，使用x射线自由电子激光器（XFELs）的连续飞秒晶体学（SFX）已经稳步发展，但实验相位仍然具有挑战性。在这里，利用来自于7 - kev（1.771埃）的来自于sprin -8埃紧凑自由电子激光器（SACLA）的x射线脉冲，在SFX体制下对模型膜蛋白细菌紫质（bR）进行了碘单波长异常衍射（SAD）、单同构置换（SIR）和单同构置换异常散射（SIRAS）相位分析。用碘标记的洗洁剂重原子添加剂13a （HAD13a）衍生出生长在双筒体中的晶体，该添加剂含有具有三个碘原子的神奇三角形I3C头基。烷基尾是洗涤剂与bR表面结合的关键。来自HAD13a的强异常和同构差分信号使来自原生晶体和衍生晶体的3000和4000个索引图像分别使用高达2.1分辨率的反射成功进行相位。当合并更多图像时，可以将数据截断为3.3埃分辨率，这是SFX相位报道案例中最低的分辨率。此外，初步的SFX实验表明，HAD13a成功衍生了G蛋白偶联的A2a腺苷受体，结晶为脂质立方相。这些结果为重新确定膜蛋白的结构铺平了道路，即使在最亮的XFEL光束下，膜蛋白的衍射也很差。
The 3D structure determination of biological macromolecules by X-ray crystallography suffers from a phase problem: to perform Fourier transformation to calculate real space density maps, both intensities and phases of structure factors are necessary; however, measured diffraction patterns give only intensities. Although serial femtosecond crystallography (SFX) using X-ray free electron lasers (XFELs) has been steadily developed since 2009, experimental phasing still remains challenging. Here, using 7.0-keV (1.771 angstrom)X-ray pulses from the SPring-8 Angstrom Compact Free Electron Laser (SACLA), iodine single-wavelength anomalous diffraction (SAD), single isomorphous replacement (SIR), and single isomorphous replacement with anomalous scattering (SIRAS) phasing were performed in an SFX regime for a model membrane protein bacteriorhodopsin (bR). The crystals grown in bicelles were derivatized with an iodine-labeled detergent heavy-atom additive 13a (HAD13a), which contains the magic triangle, I3C head group with three iodine atoms. The alkyl tail was essential for binding of the detergent to the surface of bR. Strong anomalous and isomorphous difference signals from HAD13a enabled successful phasing using reflections up to 2.1- resolution from on 3,000 and 4,000 indexed images from native and derivative crystals, respectively. When more images were merged, structure solution was possible with data truncated at 3.3-angstrom resoluti, which is the lowest resolution among the reported cases of SFX phasing. Moreover, preliminary SFX experiment showed that HAD13a successfully derivatized the G protein-coupled A2a adenosine receptor crystallized in lipidic cubic phases. These results pave the way for de novo structure determination of membrane proteins, which often diffract poorly, even with the brightest XFEL beams.