Structural Determination of Gi-scFv16 Complex

For G_i1/scFv16 crystallization, separately purified and concentrated Gα_i1, Gβ1γ2C68S and scFv16 were mixed at a 1:1:1.2 molar ratio and incubated for 30 min at 24 °C. The resulting G_i1/scFv16 complex was purified from uncomplexed subunits and free scFv16 by SEC in the buffer containing 20 mM Hepes pH7.5, 100 mM NaCl, 1 mM MgCl2, 10 μM GDP and 100 μM TCEP. Purified G_i1/scFv16 was incubated with 1 mM aluminium chloride and 50 mM sodium fluoride for 1 h on ice, concentrated to 10–15 mg/mL and crystallized using the hanging drop vapour diffusion method at 20 °C against a reservoir solution containing 10% PEG 8000, 0.1 M Sodium citrate pH 5.0, 1 mM MgCl₂, 10 μM GDP, 100 μM TCEP, 1 mM aluminium chloride and 10 mM sodium fluoride. Crystals appeared within a few hours and grew to the full size in 5 days. Crystals were soaked into the reservoir solution supplemented with 25% glycerol as a cryo-protectant and flash frozen in liquid nitrogen. The X-ray data set was collected at the experimental station 12-2 in the Stanford Synchrotron Radiation Lightsource. Diffraction data were integrated by XDS^{36 (link)}, scaled and merged by AIMLESS^{37 (link)}. The structure was solved by the molecular replacement in Phaser^{38 (link)} using the heterotrimeric Gi-protein (1GP2) and scFv fragment (4NKD) as independent search models. Manual model building was performed in Coot^{39 (link)} and refinement was performed with Phenix refine^{40 (link),41 (link)}. Ramachandran statistics are favoured 96.6%, allowed 3.4%, outlier 0.0%.