We generated three full-atom homology models of the α2β3γ2 GABAA receptor using the Rosetta molecular modeling suite (Rohl et al., 2004) with membrane environment–specific energy functions (Yarov-Yarovoy et al., 2012). A model of the open state was based upon the X-ray structure of the β3-extracellular domain (ECD)–α5-transmembrane domain (TMD) chimera, which had been reported in the presence and absence of the neurosteroid pregnanolone (Miller et al., 2017). The pregnanolone-bound structure was chosen as a template [Protein Data Bank (pdb): 5O8F]. Two additional models were generated based on the cryo-EM structure of α1β3γ2 (Masiulis et al., 2019) with alprazolam (pdb: 6HUO) or with picrotoxinin bound (pdb: 6HUG). Before homology modeling, all ligands and nanobodies were removed from the templates, and Jalview 2 (http://www.jalview.org/) was used to align the sequences of α2, β3, and γ2 with the templates. For the first model based on the β3ECD-α5TMD chimera, the ECDs of α2, β3, and γ2 were aligned with the β3ECD, and the TMDs were aligned with the α5TMD. The sequence homology between α2 and α5 or α1 is 73% and 70% in the TMD, respectively. The sequence homology between β3 and α2 or γ2 is 33% in the ECD. No additional loop modeling was performed since we were primarily interested in the well resolved TMD domains. All three homology models were refined using RosettaES (Frenz et al., 2017). Ten thousand models were generated, and the top-10 converging, lowest-energy models were selected and subjected to a final round of side-chain relaxation to minimize the energy. Before transferring the models to RosettaLigand, Glide, or Swissdock for ligand docking, post-translational modified amino acids (e.g., glycosylated or palmitoylated) were converted to standard amino acids to avoid problems with minimization procedures in these programs. Verification of correct residues was performed visually. As described below, we then first probed the TMD with two ligands (picrotoxinin and EBOB) known to bind to the NCA site to verify how suitable our models were for proceeding to dock TETS.

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