BRIL-NOP was expressed in Spodoptera frugiperda (Sf9) insect cells. Ligand binding asays were performed as described in Methods online. Sf9 membranes were solubilized using 0.5% n-dodecyl-β-D-maltopyranoside (w/v) and 0.1% cholesteryl hemisuccinate (w/v), and purified by immobilized metal ion affinity chromatography (IMAC). Receptor crystallization was performed by the lipidic cubic phase (LCP) method. The protein-LCP mixture contained 40% (w/w) concentrated receptor solution, 54% (w/w) monoolein, and 6% (w/w) cholesterol. Crystals were grown in 40 nL protein-laden LCP bolus overlaid by 0.8 μL of precipitant solution (25–30% (v/v) PEG 400, 100–200 mM potassium sodium tartrate tetrahydrate, 100 mM BIS-TRIS propane [pH 6.4]) at 20 °C. Crystals were harvested directly from LCP matrix and flash frozen in liquid nitrogen. X-ray diffraction data were collected at 100 K on the 23ID-B/D beamline (GM/CA CAT) of the Advanced Photon Source at the Argonne National Laboratory using a 10 μm collimated minibeam. Diffraction data from 23 crystals were merged for the final dataset. Data collection, processing, structure solution and refinement are described in Methods online.
Full Methods and any associated references are available in the online version of the paper at (web).