Binding of agonist IXO and Gi protein mimetic nanobody Nb9–8 to the M2 muscarinic GPCR was captured in one of five GaMD simulations: (A) Trajectories of a nitrogen atom in IXO (beads) and the β8 strand of Nb9–8 (ribbons) colored by simulation time in a blue (0 ns)–white (2250 ns)–red (4500 ns) scale. (B) RMSDs of the IXO and Nb9–8 relative to the X-ray structure, Tyr1043.33-Tyr4036.51-Tyr4267.39 triangle perimeter and Arg1213.50-Thr3866.34 distance calculated from the simulation. Dashed lines indicate X-ray structural values of the M2 receptor (3UON: green and 4MQS: red). (C) Binding pose of IXO (spheres) in the receptor extracellular vestibule with 13.84 Å RMSD relative to the X-ray conformation (yellow spheres). Residues found within 5 Å of IXO are highlighted in sticks. (D) Binding of Nb9–8 (cyan), which exhibits only 2.48 Å RMSD in the protein core (the β2, β3, β6, β7 and β8 strands). X-ray conformations of the M2 receptor and nanobody are shown in orange and purple ribbons, respectively. Adapted with permission from Miao et al. (2018). https://www.pnas.org/content/115/12/3036. Copyright 2018 National Academy of Sciences.