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.