Fig. 4: Ligand-specific activation trajectories and equilibria of M₂R–G-protein signalling complexes.

Schematic three-dimensional representation of the time-resolved, agonist-mediated formation of GPCR signalling complexes in intact cells. The planes illustrate the receptor’s extracellular surface, and the numbered positions indicate the six biosensors that were sensitive to G-protein modulation (Fig. 3). Agonist-promoted changes in fluorescence are shown as peaks. The mean fluorescence changes (Fig. 2) define the height of each peak. For better visualization, the direction of fluorescence changes was inverted (that is, fluorescence decreases are shown as increasing peaks). Peaks from the same group of biosensors are shown in the same colour. The colour of each plane is the sum of the colours of all peaks in that plane. This representation yields unique colours that enable visual discrimination between ligand-specific receptor–G protein signalling complexes. For simplification, the apo state is depicted in grey and does not result from the sum of peak colours. The positions of the conformational equilibria at steady state are indicated by the equilibrium arrows. Indicated times are apparent on-rates of agonist-specific fluorescence changes (Extended Data Fig. 9 and Supplementary Table 3). The superscript hash symbols indicate intermediate complexes. The C in the planes stands for complex.