Fig. 1: Structural and functional analysis of ligand-receptor interactions at the M₅ mAChR.

a ML380-ACh interaction in IP1 accumulation assay at WT M₅ mAChR (left) or M₅ EH4 pocket mutant (right) in CHO cells. Data points are mean ± SEM values from 7 (WT M₅ mAChR) and 3 (M₅ EH4 mutant) experiments performed in duplicate. An operational model of allosterism was fit to the grouped data. b Effects of the M₅ mAChR mutations on the pK_B of ML380. Data shown are the mean ± SEM of the affinity estimates derived from a global least-squares fit of an allosteric model to three independent experiments for all conditions, except for WT M₅ mAChR (n = 7). Pharmacology parameters were shared across experiments to yield a single best estimate of each parameter and its associated standard error, as derived from the nonlinear regression algorithm. This global pooled analysis approach ensured model convergence in all instances. *, significantly different from WT, p < 0.05, one-way ANOVA, Dunnett’s post hoc test. Parameters obtained are listed in Supplementary Table 1. c M₅ mAChR mutated residues shown on receptor structure. d EH4 pocket residues are shown as purple sticks. e ECV residues are shown as red sticks. f Consensus cryo-EM map of M₅ mAChR-mGα_q/Gβ₁γ₂ complex with iperoxo at 2.8 Å (FSC 0.143). Model colouring: dark blue (receptor), red (mGα_q), green (Gβ₁), yellow (Gγ₁). g Cryo-EM density for iperoxo in orthosteric site (local refined map, contour level 0.36). h Iperoxo-orthosteric site interactions. Interaction colouring: pink (charge-charge); black (hydrogen bonds); purple (cation-π). Source data are provided as a Source Data file.