Extended Data 2: Structural and electrophysiological analyses of human α1β3γ2L GABAA receptor in complex with PTX and GABA.
From: GABAA receptor signalling mechanisms revealed by structural pharmacology
a, FSC curves for the 3D reconstruction of the GABAA receptor bound to PTX and GABA. Curves are shown for the phase randomization, unmasked, masked and phase-randomization-corrected masked maps. b, Validation of the model refinement protocol. Curves are shown for model versus summed map (FSCfull), model refined in half-map 1 versus half-map 1 (FSCwork) and model refined in half-map 1 versus half-map 2 (FSCfree). c, The final, unsharpened cryo-EM map coloured by local resolution (estimated using MonoRes61). d, Superposition of the PTX-bound and PTX/GABA-bound α1β3γ2 receptor based on the global TMD alignment. The GABA-induced movements of loop-C in each of the β3 subunits are highlighted by green lines between Cα atoms of Thr202 residues. GABA is shown as spheres (carbon, khaki; nitrogen, blue; oxygen, red). e, Cryo-EM density segments showing GABA-binding sites in the PTX/GABA-bound structure. f–h, Representative whole-cell current traces elicited from the same HEK293 cell by three 8.8-s pulses of GABA (5 μM) plus Mb38 (2 μM), each separated by a 1-min wash: control (f); one second after the start of the second 8.8-s pulse, PTX (800 μM) was co-applied for 4 s (g); wash control showing full recovery (h). PTX inhibited currents by 106 ± 2.6% (mean ± s.d.; n = 6 cells). In addition, the protocol was repeated with outside-out patches (117 ± 9% (mean ± s.d.); n = 5 patches). i, Globally superposed PTX-bound and PTX/GABA-bound α1β3γ2 receptor transmembrane domains viewed from the extracellular space. Side chains of 9ʹ Leu residues are shown as sticks, whereas PTX is represented as balls and sticks. j, k, Superposition of α1 subunit ECDs from PTX-bound and PTX/GABA-bound α1β3γ2 receptors reveal the relative β3 ECD motions towards α1 ECDs, as viewed from outside of the receptor (j) and from the vestibule (k). Differences in distances (Å) between the selected Cα atoms in the complexes without and with GABA are indicated by lines. The PTX-bound structure is shown in grey and the PTX/GABA-bound structure is coloured by subunit (α1, red; β3, blue; γ2, yellow).
