Fig. 5: The designed Hyd_high7 structure adopts an unforeseen activated conformation.

a, Backbone representation of the designed Hyd_high7 structure bound to the ligand agonist CGS21680 and N-terminally fused to a T4Lcp variant. b, Cytoplasmic view of the following superimposed structures: antagonist-bound A2AR (PDB: 3EML, yellow), agonist-bound, computationally designed Hyd_high7 (green), Gt peptide-bound opsin active state (PDB: 3DQB, blue), and agonist- and mini-Gs-bound A2AR thermostabilized by alanine scanning (PDB: 5G53, magenta). The structures are ranked according to the magnitude of the conformational changes towards the fully active state (back arrow). c–e, Conformations of key conserved microswitches involved in receptor activation (in addition to the reference structures described in panel b, the following structure is also represented: agonist-bound A2AR thermostabilized by alanine scanning (PDB: 2YDO, grey)): the TMH3–TMH5–TMH6 interface around the W6.48 toggle switch in the receptor TM core (c), the TM3–TMH5–TMH6 interface around the intracellular DR^3.50Y motif (d) and the TMH6–TMH7 interface around the intracellular N^7.49PXXY^7.53 motif (e). Conformational changes between inactive, partially active and fully active states are indicated by distinct black arrows. f–k, Comparison of the conformation of the designed (black) and neighbouring native (grey) residue microswitches in the agonist-bound designed Hyd_high7 structure (f) and the agonist- and Gs-bound Hyd_high7 design model (g) as well as the TMH5–TMH6 core interface (h), the TMH6–TMH7 intracellular interface with bound Gs (magenta) (i), the TMH1–TMH2–TMH3 core interface at the Na⁺ ion-conserved binding site (j) and the TMH3–TMH6–TMH7 intracellular interface (k).