1 oleoyl rac glycerol monoolein

PAM, OLA, LNA, ARA, EPA, DHA, L-α-phosphatidylcholine, cholesterol, sodium taurocholate, 1-oleoyl-rac-glycerol (monoolein), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) sodium salt 99%, non-essential amino acids, lucifer yellow, dimethyl sulfoxide (DMSO) and all Hank’s Balanced Salt Solution (HBSS) buffer constituents were purchased from Sigma-Aldrich (Bornem, Belgium). Cholestyramine was purchased from Sequoia Research Products Ltd. (Pangbourne, UK). [1, 2-³H (N)]-cholesterol (1.85 TBq/mmol) was purchased from Perkin Elmer (NEN, USA). [¹⁴C]-sodium taurocholate (1.89 Gbq/mmol) was purchased from Amersham International (Buckinghamshire, United Kingdom).
Caco-2 cells were purchased from American Tissue Culture Collection (Rockville, USA). Dulbecco’s modified Eagle medium (DMEM), fetal bovine serum (FBS), 100 × nonessential amino acids, 100 × penicillin and streptomycin, 0.25% trypsin with ethylenediaminetetraacetic acid (EDTA) and BSA (Bovine serum albumin) were purchased from Thermo Scientific HyClone (Logan, USA). Transwell permeable polycarbonate inserts (0.4 μm) and 12-well cell culture plates were obtained from Corning Costar (New York, USA). Primers used in quantification of mRNA by PCR were provided by Sango Biotech (Shanghai, China). Rabbit NPC1L1 monoclonal antibody was purchased from Epitomics (5386-1, California, USA).

The purified HwBR protein was concentrated to ∼17 mg/ml, as estimated by ultraviolet absorbance, and it was mixed with 1-oleoyl-rac-glycerol (monoolein; Sigma-Aldrich) at a 2:3 (w/w) protein-to-lipid ratio using the twin-syringe mixing method. The volume of each drop was 0.2 μl of protein-lipid mixture plus 1 μl. HwBR crystals of the trimeric form were grown in 0.05 m sodium citrate, pH 5.5, 0.05 m NaCl, and 15% (v/v) PEG 400, and antiparallel dimeric crystals were grown in 0.1 m ammonium sulfate, 0.1 m sodium chloride, 0.01 m sodium acetate, pH 4.0, and 16.5% (v/v) PEG 200. The size of the crystals reached about 50 × 50 × 5 μm within 2–30 days at 20 °C. The HwBR trimeric form crystals were soaked in 30% (v/v) glycerol as a cryoprotectant before harvest.
X-ray diffraction data were collected at BL15A1 of the National Synchrotron Radiation Research Center (NSRRC), Hsinchu, Taiwan and at 44XU of SPring-8, Sayo, Japan. The data were processed using HKL2000 (53 ). We obtained the phases by molecular replacement using archaerhodopsin-2 as a template (1VGO) (25 (link)). The PHENIX (38 (link)), refmac5 (39 (link)), and COOT (40 (link)) programs were used for molecular replacement, structural refinement, and structural viewing, respectively. All structure figures were prepared in PyMOL (Schrödinger, LLC).