Abstract G Protein-Coupled Receptors (GPCRs), or 7-transmembrane receptors, are a superfamily of membrane proteins that are critically important to physiological processes in the human body. Determining high-resolution structures of GPCRs without signaling partners bound requires crystallization in lipidic cubic phase (LCP). GPCR crystals grown in LCP are often too small for traditional X-ray crystallography. These microcrystals are ideal for investigation by microcrystal electron diffraction (MicroED), but the gel-like nature of LCP makes traditional approaches to MicroED sample preparation insurmountable. Here we show that the structure of a human A 2A adenosine receptor can be determined by MicroED after converting the LCP into the sponge phase followed by cryoFIB milling. We determined the structure of the A 2A receptor to 2.8 Å resolution and resolved an antagonist in its orthosteric ligand-binding site as well as 4 cholesterol molecules bound to the receptor. This study lays the groundwork for future GPCR structural studies using single microcrystals that would otherwise be impossible by other crystallographic methods. One sentence summary FIB milled LCP-GPCR structure determined by MicroED

MicroED structure of the human adenosine receptor determined from a single nanocrystal in LCP