Extended Data Fig. 6: Molecular dynamics simulations of inactive (apo) and active (agonist- and PAM-bound) states.

a, VFT fluctuations in the inactive state are illustrated by comparing molecular dynamics snapshots at 500 ns for all 5 trajectories (cartoon, purple for α-helices and yellow for β-strands) against the cryo-EM structure (grey surface). Cyan spheres indicate nitrogen of hydrophilic heads of the POPC lipid bilayer. Asterisk corresponds to trajectory 1. b, Traces of Cα r.m.s.d. for different domains in trajectory 1. The structures in the trajectory were aligned by TMDs against the cryo-EM structure. c, Distribution of distances for ionic interactions between TM3 and TM5 at the intracellular side of the apo GABA_B, as observed in molecular dynamics simulations. d, e, Membrane-coupled ANM analysis of the inactive (d)- and active (e)-state cryo-EM structures. Eigenvectors of the slowest mode are drawn on every eighth Cα as magenta arrows. In the inset heat map, overall dynamics of the protein as observed in molecular dynamics simulations are compared against that predicted from ANM analysis on electron microscopy structures; correlations between eigenvectors of the ten slowest ANM modes and the principal components of Cα motions in molecular dynamics simulations are calculated, showing concurrence between the two. f, Distance plots as observed in agonist- and PAM-bound trajectories for interactions between SKF97541 (SKF) and various contact residues in the GB1 pocket (left column), interactions between GS39783 (PAM) and its contact residues at the TMD interface (right column, rows 1–3) and interactions between GB1 and GB2 residues (right column, rows 4 and 5). g, h, Traces of Cα r.m.s.d. on the overall structure for individual molecular dynamics trajectories (green) and plots of root mean square fluctuations (r.m.s.f.) per residue in GB1 (blue) and GB2 (yellow) subunits as observed in molecular dynamics simulations for inactive (g) and active (h) states. i, j, Stacked snapshots of GS39783 (i) and SKF97541 (j) binding site for one of the trajectories, taken every 50 ns and spanning 500 ns. In traces of Cα r.m.s.d. (b, g, h), data (solid line) are presented as an average within a sliding window of 500 ps; shading refers to 95% confidence interval (n = 500). In plots of r.m.s.f. (g, h), data (solid line) are shown as an average of 5 independent trajectories at each residue position; shading refers to 95% confidence interval (n = 5).