hKOR-T4L was expressed in Spodoptera frugiperda (Sf9) cells. Ligand-binding and functional assays were performed as described in Methods. Sf9 cells were solubilized using 1% (w/v) n-dodecyl-β-D-maltopyranoside (DDM) and 0.2% (w/v) cholesteryl hemisuccinate (CHS), and purified by immobilized metal ion affinity chromatography (IMAC), followed by reverse IMAC after cleaving N-terminal FLAG-10xHis tags by His-tagged Tobacco Etch Virus (TEV) protease. The purified protein was mixed with monoolein and cholesterol in a ratio of 40%:54%:6% (w/w) to form lipidic cubic phase (LCP) from which the receptor was crystallized. Crystals were grown at 20 °C in 45 nl protein-laden LCP boluses overlaid by 800 nl of precipitant solutions as described in Methods. Crystals were harvested from the LCP matrix and flash frozen in liquid nitrogen. X-ray diffraction data were collected on the 23ID-B/D beamline (GM/CA CAT) at the Advanced Photon Source, Argonne, IL using a 10 μm minibeam at wavelength of 1.0330 Å. Data collection, processing, structure solution and refinement are described in Methods. Modeling of JDTic analogues and hKOR-selective morphine derivatives nor-BNI and GNTI was performed using ICM-Pro; SYBYL-X 1.3 and GOLD Suite 5.1 were used to model RB-64 complexes, as described in Methods.
Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.