Strategic advances for cryo-EM structural studies of small (<100 kDa) GPCRs

Membrane proteins constitute ~25-30% of the human proteome, with G protein-coupled receptors (GPCRs) representing the largest family and targets of ~35% of FDA-approved drugs. While cryo-electron microscopy (cryo-EM) has transformed GPCR structural biology, resolving small GPCR complexes, particularly those in the apo and inactive states, remains challenging due to their limited size and conformational flexibility. This limitation is significant, as many therapeutics act by stabilizing inactive GPCR conformations. In this review, we discuss the key obstacles to visualizing apo/inactive GPCRs by cryo-EM, summarize fusion and target-binding strategies that have enabled their structural determination, outline practical considerations for fiducial design, and highlight emerging AI-driven approaches likely to further expand the scope of GPCR structural studies.