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

总结 该项目的总体目标是使用最近开发的低温电子显微镜技术， 微晶电子衍射（MicroED）用于G蛋白偶联物的结构测定 受体（GPCR）。GPCR是一类非常重要的膜蛋白， 用于控制各种生理反应。由于其重要的生理作用， 目前批准的大部分药物靶向这些受体， 药物开发的目标。尽管GPCR很重要，但详细了解其高 分辨率的结构和功能在很大程度上是有限的，这是因为与增长相关的困难 X射线晶体学所需的大晶体。在本项目中，MicroED将用于研究GPCR 结构，因为该方法能够从几个数量级的微晶确定结构， 这比传统的X射线晶体学所使用的要小。该项目将雇用和 优化新的MicroED样品制备方法，以便从GPCR收集数据 微晶生长在粘性无规立方相（LCP）。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 在Aim 1中使用先前解决的β-2肾上腺素能受体（β2AR）和A2 A腺苷进行了验证 受体（A2 AAR）作为模型。新的结构细节将在目标2和3中通过使用MicroED进行研究 提高视紫红质-抑制蛋白复合物的分辨率和建模（目标2），最后， 确定5-羟色胺受体5 HT 4的新结构（目的3）。本项目的长期目标 是开发和使用MicroED作为GPCR的高通量结构测定方法， 其他重要的膜蛋白在LCP中生长。通过确定这些目标中概述的结构， 不仅将对GPCR的结构和功能有新的了解，而且优化的方案将 为多种膜蛋白样品的MicroED分析打开了大门。这将使MicroED 一个有价值的工具，用于确定膜蛋白结构的目标，已经抵制其他 结构方法，因为优化X射线晶体学的晶体尺寸的困难。

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.