Swiss Light Source, Paul Scherrer Institut, Forschungsstrasse 111, Villigen-PSI, 5232, Switzerland.
Membrane Structural and Functional Biology (MS&FB) Group, School of Medicine and School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland.
Methods Mol Biol. 2020;2127:293-319. doi: 10.1007/978-1-0716-0373-4_20.
The lipid cubic phases (LCP) have enabled the determination of many important high-resolution structures of membrane proteins such as G-protein-coupled receptors, photosensitive proteins, enzymes, channels, and transporters. However, harvesting the crystals from the glass or plastic plates in which crystals grow is challenging. The in meso in situ serial X-ray crystallography (IMISX) method uses thin plastic windowed plates that minimize LCP crystal manipulation. The method, which is compatible with high-throughput in situ measurements, allows systematic diffraction screening and rapid data collection from hundreds of microcrystals in in meso crystallization wells without direct crystal harvesting. In this chapter, we describe an IMISX protocol for in situ serial X-ray data collection of LCP-grown crystals at both cryogenic and room temperatures which includes the crystallization setup, sample delivery, automated serial diffraction data collection, and experimental phasing. We also detail how the IMISX method was applied successfully for the structure determination of two novel targets-the undecaprenyl-pyrophosphate phosphatase BacA and the chemokine G-protein-coupled receptor CCR2A.
脂质立方相 (LCP) 使得许多重要的膜蛋白的高分辨率结构得以确定，如 G 蛋白偶联受体、光敏蛋白、酶、通道和转运蛋白。然而，从玻璃或塑料板中收获晶体是具有挑战性的。中相原位连续 X 射线晶体学 (IMISX) 方法使用薄的带窗塑料板，最大限度地减少 LCP 晶体的操作。该方法与高通量原位测量兼容，允许从中相结晶井中的数百个微晶体中进行系统的衍射筛选和快速数据收集，而无需直接晶体收获。在本章中，我们描述了一个在低温和室温下进行 LCP 生长晶体的原位连续 X 射线数据收集的 IMISX 方案，包括结晶设置、样品传递、自动化连续衍射数据收集和实验相位确定。我们还详细介绍了如何成功地将 IMISX 方法应用于两个新靶标的结构确定，即十一碳烯焦磷酸磷酸酶 BacA 和趋化因子 G 蛋白偶联受体 CCR2A。
