Fig. 3. Correlation between ligand efficacy and amide-I band ratio.

a Efficacies of the different M₂R ligands toward G_i activation in the NanoBiT G-protein dissociation assay. E_max values were calculated from the concentration-response sigmoidal curves in Extended Data Fig. 6 and were normalized to that of ACh performed in parallel. Bars and error bars represent mean and SEM, respectively, of 5 or 10 (shown in parenthesis) independent experiments with each performed in duplicate. b Correlation between ligand efficacy and the relative intensities of the amide-I bands. The relative intensities of the amide-I bands are calculated by the equation of efficacy rate, which is derived from Fig. 2c. Ligand efficacy is determined by the NanoBiT G-protein dissociation assay from a. Agonists (ACh, Meta, Are, and Carb) are shown as red circles, partial agonists (Pilo, McN, and Xano) as orange circles, and antagonists (Atro, Scop, and Ipra) as purple circles.