Structure Determination of G Protein-Coupled Receptors by Microcrystal Electron Diffraction

摘要 这个项目的总体目标是使用最近开发的冷冻电子显微镜技术 微晶电子衍射法测定G蛋白偶联产物的结构 受体(GPCR)。GPCRs是一类极其重要的膜蛋白，负责 来控制各种各样的生理反应。由于它们的关键生理作用，一个 目前批准的药物中有很大一部分针对这些受体，它们代表着诱人的 药物开发的目标。尽管GPCRs很重要，但详细了解其高 分辨率结构和功能在很大程度上受到相关生长困难的限制 X射线结晶学所必需的大晶体。在本项目中，将使用MicroED来研究GPCR 结构，因为该方法能够从微晶体中确定几个数量级的结构 比传统的X射线结晶学所用的要小。该项目将雇用和 优化新的MicroED样品制备方法，允许从GPCR收集数据 在粘性脂立方相(LCP)中生长微晶。将收集电子衍射数据 并使用先前开发的MicroED方法进行处理。GPCR的新方法将是 使用先前解决的β-2肾上腺素能受体(β2AR)和A2A腺苷在AIM 1中进行验证 受体(A2AAR)作为模型。在AIMS 2和AIMS 3中将使用MicroED研究新的结构细节 提高视紫红质-arrestin复合体的分辨率和模型化(目标2)，最后 确定5-羟色胺受体5HT4的新结构(目标3)。这个项目的长期目标是 是开发和使用MicroED作为GPCRs和GPCRs的高通量结构测定方法 LCP中生长的其他重要的膜蛋白。通过确定这些目标中概述的结构， 不仅将揭示GPCR的结构和功能，而且优化后的协议将 为各种膜蛋白样品的MicroED分析打开大门。这将使MicroED 膜蛋白结构测定的有价值的工具 结构方法，因为在优化X射线结晶学的晶体尺寸方面存在困难。

SUMMARY The overall aim of this project is to use the recently developed cryo-electron microscopy technique of microcrystal electron diffraction (MicroED) for the structure determination of G protein-coupled receptors (GPCR). GPCRs are an extremely important class of membrane proteins and are responsible for controlling a wide variety of physiological responses. Because of their key physiological roles, a large percentage of currently approved drugs target these receptors, and they represent attractive targets for drug development. Despite the importance of GPCRs, detailed understanding of their high resolution structure and function is limited in large part because of the difficulty associated growing large crystals necessary for X-ray crystallography. In this project, MicroED will be used to study GPCR structure, as the method is capable of determining structures from microcrystals several orders of magnitude smaller than those used by conventional X-ray crystallography. The project will employ and optimize new MicroED sample preparation methodology to allow data collection from GPCR microcrystals grown in the viscous lipidic cubic phase (LCP). Electron diffraction data will be collected and processed using previously developed MicroED methods. The new methods for GPCRs will be validated in Aim 1 using the previously solved beta-2 adrenergic receptor (β2AR) and A2A Adenosine receptor (A2AAR) as models. New structural details will be studied in Aims 2 and 3 by using MicroED to improve the resolution and modeling of the rhodopsin-arrestin complex (Aim 2), and finally to determine a novel structure of the serotonin receptor 5HT4 (Aim 3). The long term goal of this project is to develop and use MicroED as a high-throughput structure determination method for GPCRs and other important membrane proteins grown in LCP. By determining the structures outlined in these aims, not only will new light will be shed on GPCR structure and function, but the optimized protocols will open the door to MicroED analysis for a variety of membrane protein samples. This will make MicroED a valuable tool for membrane protein structure determination of targets that have resisted other structural methods because of difficulties with optimizing crystal size for X-ray crystallography.