Universal pipeline for high-resolution GPCR structure determination

G protein-coupled receptors (GPCRs) regulate human physiology and are major drug targets. Although cryo-electron microscopy has accelerated GPCR structural biology, inactive-state structures remain difficult because current fusion-based strategies often require extensive experimental screening to identify rigid constructs suitable for high-resolution reconstruction. Here we introduce a universal pipeline that integrates an in silico fusion construct screening program, NOAH (nonexperimental, artificial-intelligence-assisted, high-throughput construct screening for structural analysis), with a de novo designed fusion protein, ARK1 (artificially designed fiducial marker). NOAH enabled structure determination of vasopressin V2 receptor bound to the antagonist tolvaptan or partial agonist OPC51803 and bradykinin B2 receptor bound to the antagonist icatibant, revealing receptor activation and inhibition mechanisms. Coupling NOAH to ARK1 improved the V2 receptor-tolvaptan map and enabled high-resolution structures of lysophosphatidic acid receptor 2 bound to Ki16425 and free fatty acid receptor 2 bound to GLPG0974. NOAH-ARK1 minimizes trial-and-error construct optimization and provides a broadly applicable route for GPCR structural analysis and drug discovery.