Tre mre

For purification, cells were thawed in 20 mM Hepes (pH 7.4), 150 mM NaCl, 10 mM MgCl₂, and CaCl₂ with Protease Inhibitor Cocktail (TargetMol). For TRE/MRE-IP-Gα_s-Gβ-Gγ complexes, 10 μM TRE/MRE (MedChemExpress) and 2 mg of Nb35 were added and incubated at room temperature for 1 hour. Membranes were solubilized with 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG; Anatrace) and 0.1% (w/v) cholesteryl hemisuccinate (CHS; Anatrace) at 4°C for 2 hours and then centrifuged at 70,000g for 30 min to remove insoluble material. Solubilized complexes were immobilized on nitrilotriacetic acid (Ni-NTA) resin (GenScript), washed with 30 column volumes of wash buffer, and eluted with 300 mM imidazole. Complexes were concentrated using 100-kDa Amicon filters and further purified by size exclusion chromatography on a Superdex 6 Increase 10/300 GL column (GE Healthcare) pre-equilibrated with size buffer containing 20 mM Hepes (pH 7.4),150 mM NaCl, 0.00075% (w/v) LMNG, 0.00025% (w/v) glyco-diosgenin [(GDN) Anatrace], 0.00025% digitonin (w/v), 0.00015% CHS, and 10 μM ligand to separate complexes. Eluted fractions were analyzed by SDS–polyacrylamide gel electrophoresis, and those containing receptor-G_s complexes were pooled, concentrated, and used for cryo-EM.