Tools to investigate class B1 GPCR conformational landscapes with cryo-EM and beyond

G protein-coupled receptors (GPCRs) are membrane proteins that act as signal transducers across cell membranes. Class B1 GPCRs, a subset of 15 receptors activated by peptide hormones, are involved in important physiological processes and diseases, making them a popular target for drug development. GPCRs are dynamic proteins and can adopt a myriad of conformational states, allowing them to bind and activate multiple intracellular signal transducers, including G proteins and β-arrestins, though all class B1 GPCRs primarily couple to the stimulatory G protein (Gs). Cryogenic electron microscopy (cryo-EM) structures of all class B1 GPCRs bound to Gs are available and provide meaningful insights into receptor function. However, there is a dearth of structural information on class B1 GPCRs in inactive and intermediate states or bound to other signal transducers, meaning we are currently only afforded a small vista into the conformational landscape these GPCRs sample. As cryo-EM-based 3D reconstructions are heavily dependent on protein stability and conformational homogeneity, currently available structures are largely limited to only those most stable conformations (i.e. Gs complexes). The present review focuses on technical aspects of obtaining class B1 GPCR structures using cryo-EM and new in silico methods that allow insight into unseen GPCR conformations, revealing more structural details of the conformational landscape.