Howard Hughes Medical Institute, University of California, Los Angeles, CA, 90095, USA.
Department of Biological Chemistry, University of California, Los Angeles, CA, 90095, USA.
Nat Commun. 2023 Feb 25;14(1):1086. doi: 10.1038/s41467-023-36733-4.
Crystallizing G protein-coupled receptors (GPCRs) in lipidic cubic phase (LCP) often yields crystals suited for the cryogenic electron microscopy (cryoEM) method microcrystal electron diffraction (MicroED). However, sample preparation is challenging. Embedded crystals cannot be targeted topologically. Here, we use an integrated fluorescence light microscope (iFLM) inside of a focused ion beam and scanning electron microscope (FIB-SEM) to identify fluorescently labeled GPCR crystals. Crystals are targeted using the iFLM and LCP is milled using a plasma focused ion beam (pFIB). The optimal ion source for preparing biological lamellae is identified using standard crystals of proteinase K. Lamellae prepared using either argon or xenon produced the highest quality data and structures. MicroED data are collected from the milled lamellae and the structures are determined. This study outlines a robust approach to identify and mill membrane protein crystals for MicroED and demonstrates plasma ion-beam milling is a powerful tool for preparing biological lamellae.
在类脂立方相 (LCP) 中结晶 G 蛋白偶联受体 (GPCR) 通常会产生适合低温电子显微镜 (cryoEM) 方法微晶电子衍射 (MicroED) 的晶体。然而,样品制备具有挑战性。嵌入的晶体在拓扑上无法定位。在这里,我们在聚焦离子束和扫描电子显微镜 (FIB-SEM) 内部使用集成荧光显微镜 (iFLM) 来识别荧光标记的 GPCR 晶体。使用 iFLM 定位晶体,并使用等离子体聚焦离子束 (pFIB) 研磨 LCP。使用蛋白酶 K 的标准晶体确定了制备生物薄片的最佳离子源。使用氩气或氙气制备的薄片产生了质量最高的数据和结构。从研磨的薄片中收集 MicroED 数据并确定结构。这项研究概述了一种用于 MicroED 识别和研磨膜蛋白晶体的强大方法,并证明等离子体离子束铣削是制备生物薄片的有力工具。