Extended Data Table 2: Cryo-EM data collection, refinement and validation statistics.

	GABA (EMDB-22037) (PDB 6X3Z)	GABA+ Diazepam (EMDB-22036) (PDB 6X3X)	GABA+ Flumazenil (EMDB-22033) (PDB 6X3U)	GABA+ Picrotoxin (EMDB-22038) (PDB 6X40)
Data collection and processing
EM Facility	HMS	UTSW	UTSW	HMS
Magnification	105 K	105 K	165 K	105 K
Voltage (kV)	300	300	300	300
Electron exposure (e−/Å2)	63.79	63.04	50.28	62.91
Defocus range (μm)	−1.8 to −2.8	−1.8 to −2.8	−0.6 to −2.1	−1.4 to −2.3
Pixel size (Å)	0.825	0.833	0.84	0.825
Symmetry imposed	C1	C1	C1	C1
Initial particle images (no.)	1,705,334	2,868,814	1,072,111	1,847,538
Particle images after 2D classification	871,313	826,254	810,710	1,550,272
Final particle images (no.) (%)‡	171,838 (19.7)	297,028 (35.9)	260,276 / 62,364 (32.1 / 7.7)	165,494 (10.7)
Map resolution (Å)	3.23	2.92	3.20 / 3.49	2.86
FSC threshold	0.143	0.143	0.143	0.143
Map resolution range (Å)	2.5 – 3.9	2 – 3.5	3 – 4.5	2 – 3.2
Refinement
Initial model used (PDB code)	6U6D + 4COF	6X3Z	6X3Z	6X3Z
Model resolution (Å)	3.34	3.02	3.70	2.99
FSC threshold	0.5	0.5	0.5	0.5
Model resolution range (Å)	n.a.	n.a.	n.a.	n.a.
Map sharpening B factor (Å2)	−109	−113	−125 / −120	−94
Model composition
Non-hydrogen atoms	17,365	17,470	17,387	17,411
Protein residues	2,121	2,121	2,121	2,121
Ligands	23	29	24	26
B factors (Å2)
Protein	62.75	40.07	24.15	62.21
Ligand	66.81	46.42	31.71	60.63
R.m.s. deviations
Bond lengths (Å)	0.007	0.006	0.005	0.007
Bond angles (°)	1.069	1.024	0.998	1.092
Validation
MolProbity score	1.59 (100th %)	1.49 (100th %)	1.47 (100th %)	1.68 (100th %)
Clashscore	5.44 (100th %)	4.14 (100th %)	4.05 (100th %)	7.16 (98th %)
Poor rotamers (%)	0.69	0.53	0.32	0.42
Ramachandran plot
Favored (%)	95.77	95.82	95.91	95.82
Allowed (%)	4.23	4.18	4.09	4.18
Disallowed (%)	0.00	0.00	0.00	0.00

^‡Percent of particles in final reconstitution compared to after 2D classification