G protein-coupled receptors (GPCRs) form the largest human membrane protein family and are important therapeutic targets with ~35% of FDA approved drugs targeting GPCRs. Drug discovery programs are enhanced by structural information detailing how ligands interact with the target protein. Traditionally for GPCRs, this has relied upon structural information derived from X-ray crystallography. However, rapid advances in cryo-EM, requiring only small amounts of protein and less homogenous protein samples, have enabled high-resolution structures to be obtained and visualisation of ligand-protein interactions. Therefore, cryo-EM has become a powerful tool in structure-based drug design programs.
There are many high-resolution cryo-EM structures of active, G protein-bound, GPCR structures but few of the receptors alone in the inactive state (with no transducer proteins bound). Inactive structures of GPCRs are challenging for cryo-EM due to their small size contained mostly within the detergent micelle, as they often lack extracellular features; hindering particle alignment during image processing. Here, we present our approach to determine the cryo-EM structure of the inactive adenosine A2A receptor previously solved by x-ray crystallography (Segala et al. 2016). The A2A receptor construct contains a BRIL fusion protein that increases the molecular weight of the sample and introduces a structural feature outside of the micelle. The sample was initially resolved to ~4.5 Å. In order to improve the resolution and map quality, we used a combination of anti-BRIL antibodies and nanobodies to further increase the size and stability of the receptor (Mukherjee et al. 2020). We hope that this approach can lead to the determination of a wider range of inactive GPCR structures subsequently aiding structure-based drug design for this important class of therapeutic targets.