GoKawiilExpression and purification of G protein heterotrimer
G protein heterotrimer was expressed as described14 in Tni cells (Expression Systems; isolated from Trichoplusia ni; no validation or mycoplasma testing) with viruses for Gα, Gβγ and Ric-8a and snap frozen in liquid nitrogen for later use. To purify wild-type G protein, cell pellets were thawed and resuspended in a lysis buffer containing 20 mM HEPES pH 7.5, 1 mM EDTA, 5% glycerol, 1 mM MgCl 2 , 5 mM β-mercaptoethanol, 100 µM GDP, Pierce Universal Nuclease, and protease inhibitor cocktail, and gently stirred for 30 min at 4 °C. The lysate was then subjected to ultracentrifugation at 100,000g for 35 min, and the supernatant was discarded. Pellets were resuspended in solubilization buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 1% sodium cholate, 5% glycerol, 1 mM MgCl 2 , 5 mM β-mercaptoethanol, 100 µM GDP, Pierce Universal Nuclease, and protease inhibitor cocktail, and allowed to gently stir for 1 h at 4 °C prior to ultracentrifugation at 100,000g for 35 min. Solubilized protein was supplemented with 30 mM imidazole and incubated for 1 h with Ni-NTA beads. The beads were collected by centrifugation at 300g, packed into a column, and washed with 10 column volumes of a series of buffers containing 30 mM imidazole and 50% solubilization/50% E2 buffer, 25% solubilization/75% E2 buffer, 12.5% solubilization/87.5% E2 buffer, and 100% E2 buffer (E2 buffer contained 20 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol, 1 mM MgCl 2 , 5 mM β-mercaptoethanol, 100 µM GDP, and 0.05% lauryl maltose neopentyl glycol (LMNG)/0.005% cholesterol hemisuccinate (CHS)). Protein was eluted with 3 column volumes of buffer containing E2 buffer and 250 mM imidazole, and was incubated with 1 mg of 3C protease per 50 mg of protein with overnight dialysis at 4 °C in dialysis tubing. The next day, the Ni-NTA column was washed with 10 column volumes of buffer containing E2 and 30 mM imidazole, and overnight cleavage was loaded over the nickel and flow through was collected. The column was washed with an additional 2 column volumes of the E2 buffer and 30 mM imidazole, and flow through was collected. Protein was concentrated subjected to size-exclusion chromatography with a Superdex 200 column and a buffer containing 20 mM HEPES pH 7.5, 100 mM NaCl, 5% glycerol, 1 mM MgCl 2 , 100 µM TCEP ((Tris(2-carboxyethyl)phosphine)), 20 µM GDP, and 0.01% LMNG/0.001% CHS. Fractions were pooled, concentrated, and snap frozen in liquid nitrogen for later usage. For single-molecule experiments instead of cutting the His tag and performing reverse chromatography, the purified G protein heterotrimers were then mixed with biotinylated Tris-NTA (Sigma, supplemented with equimolar NiCl 2 ) at a molar ratio of 2:3 and incubated on ice for 1 h. The G protein heterotrimer and biotinylated Tris-Ni2+-NTA complexes were subsequently purified using size-exclusion chromatography (Superdex200, Cytiva).
Expression and purification of NTSR1
Pellets containing NTSR1 were expressed in Sf9 cells (Expression Systems, no validation or mycoplasma testing) with the baculovirus system as described12 and snap frozen in liquid nitrogen for storage. To purify NTSR1, cell pellets were thawed and lysed in hypotonic lysis buffer containing 10 mM HEPES pH 7.5, 1 mM benzamidine, Pierce Universal Nuclease, protease inhibitor cocktail, 1 mM EDTA, 1 mM MgCl 2 , and 100 µM TCEP, and gently stirred for 1 h at 4 °C. Subsequently, membranes were collected by ultracentrifugation at 100,000g, the supernatant was discarded, and pellets containing the membranes were resuspended in solubilization buffer containing 20 mM HEPES pH 7.5, 500 mM NaCl, 1 mM benzamidine, Pierce Universal Nuclease, 1 mM MgCl 2 , 100 µM TCEP, and protease inhibitor cocktail. Detergent was then added dropwise while gently stirring at 4 °C to a final concentration of 1% LMNG/0.1% CHS/0.1% sodium cholate. After 3 h of stirring, insoluble debris was removed via ultracentrifugation at 100,000g, supplemented with 20 mM imidazole, and loaded over a TALON resin column. The columns were washed with 10 column volumes of buffer containing 20 mM HEPES pH 7.5, 20 mM imidazole, 500 mM NaCl, and 0.1% LMNG/0.01% CHS. Protein was eluted with 2 column volumes of buffer containing 20 mM HEPES pH 7.5, 250 mM imidazole, 250 mM NaCl, 10% glycerol, and 0.01% LMNG/0.001% CHS. Purified receptor was concentrated and subjected to size-exclusion chromatography with a Superdex 200 column and a buffer containing 100 mM NaCl, 20 mM HEPES pH 7.5, 5% glycerol, and 0.01% LMNG/0.001% CHS. Peak fractions were pooled, concentrated, and further supplemented with 5% glycerol, then snap frozen for use for complexation.
Formation and purification of NTSR1–G protein complex
Complex formation was initiated by mixing NTS 8–13 -bound receptor (NTSR1 incubated with NTS 8–13 for 1 h) with a ~1.25 molar excess of G protein and incubating for 1 h on ice before addition of apyrase to remove GDP and HRV 3C protease to cleave the GFP tag from NTSR1 and incubation on ice overnight. Complex was then diluted in a buffer containing 100 mM NaCl, 20 mM HEPES pH 7.5, 10 μM NTS 8–13 , 0.01% LMNG/0.001% CHS, 5 mM CaCl 2 and loaded onto M1 Flag resin, washed with a buffer of 100 mM NaCl, 20 mM HEPES pH 7.5, 10 μM NTS 8–13 , 0.005% LMNG/0.0005% CHS, 5 mM CaCl 2 to remove excess G protein. NTSR1–G protein complex, and then eluted with a buffer of 100 mM NaCl, 20 mM HEPES pH 7.5, 10 μM NTS 8–13 , 0.005% LMNG/0.0005% CHS, 200 μg ml−1 Flag peptide and 1 mM EDTA. Eluted complex was concentrated and loaded onto a Superdex 200 size-exclusion column in a buffer of 100 mM NaCl, 20 mM HEPES pH 7.5, 10 μM NTS 8–13 , 0.001% LMNG/0.00033% GDN/0.0001% CHS, 2 mM MgCl 2 . Fractions of complex were concentrated to 6–10 mg ml−1 for cryo-EM studies.
Preparation of NTSR1–G protein complex in nanodisc
NTSR1–G i1 complex was formed overnight as described in detergent, and then incubated for 1 h with solution containing MSP1D1 belt protein (Sigma) mixed with 3:2 POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine):POPG (1-palmitoyl-2-oleoylphosphatidylglycerol) at a 1:5:400 ratio of complex:MSP1D1:lipid. Two 1 h incubations with Bio-Beads SM-2 were performed followed by a final 8 h incubation with fresh Bio-Beads SM-2. NTSR1–G protein complex was then further purified as described for the detergent complex with Flag and size-exclusion chromatography, but in the absence of detergent. Nanodisc formation was confirmed with SDS–PAGE and concentrated to 2–4 mg ml−1 for cryo-EM studies.
Cryo-EM sample preparation and data collection
UltrAufoil R1.2/1.3 300 mesh grids were glow discharged in a Pelco unit for 45 s at 10 mA before being used for grid freezing. 3 μl of sample was pipetted onto a grid in a vitrobot held at 100% humidity and 4 °C, 0.3 μl of 10 mM GTP was added for the time-resolved cryo-EM grids, and the grids were blotted and plunge frozen in liquid ethane with a preprogramed blot and wait time to achieve 6 s and 20 s time points, chosen based upon prior studies with β 2 AR and MOR and consistent with the lifetimes observed in single-molecule based fluorescence assays. Detergent data were collected on a G4 Titan Krios equipped with a K3 direct electron detector and a Bioquantum energy filter. Details of each data collection can be found in Extended Data Tables 1 and 2 and Supplementary Table 2, but in brief, grids were imaged with a nominal defocus range from −0.6 to −1.6 μm and a target dose of 55 e− Å−2 over 40 total frames in super-resolution mode. Nanodisc data were collected on a G3 Titan Krios equipped with a Falcion IV/Selectris energy filter, with similar parameters to the detergent datasets.
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