GoKawiilExpression and purification of β 2 AR in Sf9 cells
The β 2 AR construct PN1 was expressed and purified as previously described39,42. In brief, Sf9 cells were infected with a PN1-containing baculovirus produced using the BestBac method. Cells were then collected and resuspended in chilled lysis buffer containing 10 mM HEPES, pH 7.4, 1 mM EDTA, 1 μM alprenolol and protease inhibitors (leupeptin and benzamidine). Lysed cells were then pelleted at 18,600 rpm for 20 min and dounced to homogeneity in chilled solubilization buffer containing 20 mM HEPES pH 7.4, 350 mM NaCl, 1% n-dodecyl β-D-maltoside (DDM), 0.1% cholesteryl hemisuccinate (CHS), 2 mM MgCl 2 , 1 μM alprenolol, protease inhibitors and benzonase. After stirring for 90 min at 4 °C and centrifugation at 18,600 rpm for 30 min, 2 mM CaCl 2 was added to the soluble fraction, which was then applied to anti-Flag (DYKDDDDK) M1 immunoaffinity resin. The receptor was then washed (20 mM HEPES, pH 7.4, 350 mM NaCl, 0.1% DDM, 0.01% CHS, 2 mM CaCl 2 , leupeptin and benzamidine), eluted (20 mM HEPES, pH 7.4, 350 mM NaCl, 0.1% DDM, 0.01% CHS, 5 mM EDTA and 200 μg ml−1 Flag peptide), and further purified on Superdex 200 10/300 Increase gel filtration column equilibrated in NH buffer (20 mM, HEPES pH 7.4, 100 mM NaCl) plus 0.1% DDM and 0.01% CHS.
To produce homogeneous β 2 AR dimer, Sf9 cells expressing PN1 were resuspended at room temperature in 20 mM HEPES, pH 7.4, 150 mM NaCl, 10% glycerol, 1 μM alprenolol, protease inhibitors and 10 μM AP. After incubation for 30 min at room temperature, membrane solubilization was initiated by adding 1% lauryl maltose neopentyl glycol (LMNG) and 0.1% CHS. The purification steps followed the same protocol as for the monomeric receptor, except that 0.01% LMNG replaced DDM in all buffers, and 10 μM AP was maintained throughout the purification.
Expression and purification of heteromeric Gα s β 1 γ 2
As previously described43,44, heterotrimeric G s was expressed and purified from Trichoplusia ni Hi5 cells. In brief, two baculoviruses were generated using the BestBac method, one encoding the wild-type human Gα s subunit and the other encoding the wild-type human β 1γ2 subunits containing a histidine tag on the N terminus of the β subunit. Cells were infected with both viruses for 48 h and collected by centrifugation. The pellet was then resuspended and stirred for 30 min at 4 °C in hypotonic buffer containing 10 mM HEPES pH 7.4, 100 μM MgCl 2 , 5 mM β-mercaptoethanol, 20 μM GDP and protease inhibitors. Lysed cells were then pelleted at 18,600 rpm for 15 min and dounced to homogeneity in chilled NH buffer plus 1% sodium cholate, 0.05% DDM, 1 mM MgCl 2 , 5 mM β-mercaptoethanol, 20 μM GDP and protease inhibitors. After solubilization for 1.5 h while stirring at 4 °C and centrifugation at 18,600 rpm for 35 min, 20 mM of imidazole was added to the soluble fraction, which was then allowed to batch-bind to washed nickel-chelated Sepharose for 2 h. Pelleted resin was then loaded into a narrow column, washed with buffers containing gradually declining amounts of cholate and eluted with NH buffer plus 0.05% DDM, 1 mM MgCl 2 , 20 μM GDP, 100 μM tris(2-carboxyethyl)phosphine (TCEP) and 250 mM imidazole. Human rhinovirus 3C protease was added to cleave the histidine tag and the eluate was dialysed overnight at 4 C in 2 l of dialysis buffer (NH buffer plus 1 mM MgCl 2 , 0.05% DDM, 20 μM GDP and 100 μM TCEP). The protein solution was run through a second nickel-chelated Sepharose column, washed with dialysis buffer supplemented with 20 mM imidazole and dephosphorylated for 30 min on ice with lambda protein phosphatase, calf intestinal phosphatase and Antarctic phosphatase with 1 mM manganese chloride. The heterotrimer was further purified from excess βγ subunits using ion-exchange chromatography on the MonoQ 10/100 GL column. The sample was loaded and washed with 20 mM HEPES, pH 7.4, 1 mM MgCl 2 , 0.05% DDM, 100 μM TCEP and 20 μM GDP. Heterotrimeric G s was then eluted with a linear salt gradient from 50 mM NaCl to 500 mM NaCl.
Expression and purification of Nb60
Nb60 was expressed and purified as previously described24. In brief, Nb60 was expressed in Escherichia coli BL21(DE3) cells. The lysates were then purified on a nickel-chelated Sepharose column and subsequently on the Superdex 200 Increase 10/300 column in 20 mM HEPES, pH 7.4, and 150 mM NaCl.
Sample preparation for cryo-EM
For the sample in detergent, purified PN1 in 0.1% DDM/0.01% CHS was loaded onto anti-Flag M1 immunoaffinity resin equilibrated in the same detergent mixture supplemented with 2 mM CaCl 2 . The receptor was exchanged into a synthetic triglucoside-based detergent, TTG-T10 (ref. 45), during which the receptor bound to the resin was washed with increasing ratios of TTG-T10 to DDM, ultimately transitioning the receptor into 0.01% TTG-T10/0.001% CHS. Each detergent exchange buffer contained NH buffer with 2 mM CaCl 2 , 1 μM carazolol and 10 μM AP. The receptor was subsequently eluted in NH buffer plus 0.01% TTG-T10, 0.001% CHS, 1 μM carazolol, 10 μM AP, 5 mM EDTA and 200 μg ml−1 Flag peptide. After the detergent exchange, PN1 was incubated for 1 h with twofold molar excess of Nb60. Excess Nb60 was cleared on anti-Flag M1 immunoaffinity resin after washing with NH buffer plus 0.0006% TTG-T10, 0.00006% CHS, 1 μM carazolol, 10 μM AP and 2 mM CaCl 2 . The complex was then eluted off the resin with NH buffer plus 0.0006% TTG-T10, 0.00006% CHS, 1 μM carazolol, 10 μM AP, 5 mM EDTA and 200 μg ml−1 Flag peptide. The final sample was concentrated to over 10 mg ml−1 and used immediately for cryo-EM grid preparation.
To prepare the AP-bound β 2 AR dimer in lipid nanodiscs, the purified dimer in LMNG was reconstituted into nanodiscs following a previous protocol42 with modifications. Lipids were prepared by mixing 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC, Avanti), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-L-serine (POPS, Avanti) and cholesterol (Sigma-Aldrich) at a molar ratio of 7:2:1, followed by drying under argon and vacuum desiccation for 2 h. Lipids were resuspended in NH buffer containing 14 mM DDM at 20 mg ml−1. The receptor was diluted to 10 μM, incubated with 50 μM BI-167107 (PubChem CID: 45483813, MedChemExpress) for 10 min on ice, and then combined with membrane scaffold protein (MSP) 1E3D1 and lipids at a molar ratio of 1:2.5:100. The mixture was incubated on ice for 1 h to allow nanodisc assembly. Detergent was removed by sequential addition of semi-wet Bio-Beads SM2 (60 mg ml−1) three times over 3 h period, followed by overnight incubation at 4 °C. The next day, the Bio-Beads were removed, and empty nanodiscs were separated by M1 affinity purification. The dimeric β 2 AR in nanodiscs were eluted in buffer containing 1 μM BI, 10 μM AP, 5 mM EDTA and 200 μg ml−1 Flag peptide. The eluate was further polished by SEC in NH buffer plus 1 μM BI and 10 μM AP. Peak fractions were pooled and concentrated to 5 mg ml−1 for grid freezing.
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