HA-signal peptide-FLAG-3C-GLP-1R-3C-8×HIS13, human DNGαs46, His6-tagged human Gβ1and Gγ2 were expressed in Tni insect cells (Expression systems) using baculovirus as previously described. Cell cultures were grown in ESF 921 serum-free media (Expression Systems) to a density of 4 million cells/ml and then infected with three separate baculoviruses at a ratio of 2:2:1 for GLP-1R, MedicGLP Weight Management DNGαs and Gβ1γ2. The culture was harvested by centrifugation 60 h post-infection and the cell pellet was stored at −80 °C. Cell pellet was thawed in 20 mM HEPES pH 7.4, 50 mM NaCl, MedicGLP Weight Management 5 mM CaCls, 2 mM MgCl2 supplemented with cOmplete Protease Inhibitor Cocktail tablets (Roche) and benzonase (Merk Millipore). Complex formation was initiated by the addition of 10 μM exendin-4 or 50 μM oxyntomodulin (China Peptides), Nb35-His (10 μg/mL) and apyrase (25 mU/mL, NEB); the suspension was incubated for 1 h at room temperature. The complex was solubilized from the membrane by 0.5% (w/v) lauryl maltose neopentyl glycol (LMNG, Anatrace) supplemented with 0.03% (w/v) cholesteryl hemisuccinate (CHS, Anatrace) for 1 h at 4 °C.
Insoluble material was removed by centrifugation at 30,000 × g for 30 min and the solubilised complex was immobilised by batch binding to M1 anti-FLAG affinity resin in the presence of 5 mM CaCl2. The resin was packed into a glass column and washed with 20 column volumes of 20 mM HEPES pH 7.4, 100 mM NaCl, 2 mM MgCl2, 5 mM CaCl2, 1 μM exendin-4 or 10 μM oxyntomodulin, 0.01% (w/v) LMNG and 0.0006% (w/v) CHS before bound material was eluted in buffer containing 5 mM EGTA and 0.1 mg/mL FLAG peptide. The complex was then concentrated using an Amicon Ultra Centrifugal Filter (MWCO 100 kDa) and subjected to size-exclusion chromatography on a Superdex 200 Increase 10/300 column (GE Healthcare) that was pre-equilibrated with 20 mM HEPES pH 7.4, 100 mM NaCl, 2 mM MgCl2, 1 μM exendin-4 or 10 μM oxyntomodulin, 0.01% (w/v) LMNG and 0.0006% (w/v) CHS to separate complex from contaminants. Eluted fractions consisting of receptor and G-protein complex were pooled and concentrated to 3-5 mg/mL.
The complex samples were flash-frozen in liquid nitrogen and stored at −80 °C. Sample collected from size-exclusion chromatography was analysed by SDS-PAGE and Western blot. For SDS-PAGE, precast gradient TGX gels (Bio-Rad) were used. Gels were either stained by Instant Blue (Expedeon) or immediately transferred to PVDF membrane (Bio-Rad) at 100 V for 1 h. The proteins on the PVDF membrane were probed with two primary antibodies, rabbit anti-Gαs C-18 antibody (cat. Gαs subunit and mouse penta-His antibody (cat. EM grids (Quantifoil, Großlöbichau, Germany, 200 mesh copper R1.2/1.3) were glow discharged for 30 s in high-pressure air using Harrick plasma cleaner (Harrick, Ithaca, NY). The sample was applied on the grid in the Vitrobot chamber (FEI Vitrobot Mark IV). The chamber of Vitrobot was set to 100% humidity at 4 °C. The sample was blotted for 5 s with a blot force of 20 and then plunged into propane-ethane mixture (37% ethane and 63% propane).
Exendin-4: Data for the GLP1R:DNGs:exendin- complex was collected on a Titan Krios microscope operated at 300 kV (ThermoFisher Scientific equipped with a Gatan Quantum energy filter operating in Zero Loss mode with an energy slit with of 20 eV and a Gatan K2 Summit direct electron detector (Gatan). Movies were taken in EFTEM nanoprobe mode, with 50 µm C2 aperture and no objective aperture, at a magnified pixel size of 0.87 Å. Each movie comprised 48 frames with a total dose of 48 e−/Å2, the exposure time was 8 s with a dose rate of 7 e−/pix/s on the detector. EPU (ThermoFisher Scientific) was used to automate data collection which involved implementing beam-tilt to collect a 3 × 3 grid of holes. As the data collection was split between two different days, the data was split into 18 optics groups. Oxyntomodulin: Data for the GLP-1R:DNGs:oxyntomodulin complex was collected on a Titan Krios microscope operated at 300 kV (ThermoFisher Scientific equipped with a Gatan Quantum energy filter and a Gatan K2 Summit direct electron detector (Gatan) and a Volta Phase Plate (ThermoFisher Scientific). Movies were taken in EFTEM nanoprobe mode, with 50 µm C2 aperture, at a magnified pixel size of 1.06 Å.
Each movie comprised 50 frames with a total dose of 50 e-/Å2, the exposure time was 8 s with a dose rate of 7 e−/pix/s on the detector. Data acquisition was done using SerialEM software at −500 nm defocus44. Exendin-4: 8816 movies were collected and subjected to motion correction using motioncor247. CTF estimation was done using Gctf software48 on the non-dose-weighted micrographs. The particles were picked from dose-weighted and low-pass filtered micrographs using crYOLO automated picking routine49. The particles were extracted in RELION 3.050 using a box size of 256 pixels. 1.52M picked particles were subjected to rounds of 2D and 3D classification in order to obtain a homogenous set of projections. This led to 422k particles which were polished and had their CTF parameters re-refined in RELION. Further rounds of 2D and 3D classification yielded a final particle stack of 277.5k particles for final 3D refinement and further rounds of masked refinements to reveal details of more flexible regions of the protein.
