Monoolein

Purified protein was concentrated to between 30 and 35 mg/ml using a centrifugal concentrator (Millipore) with a 100 kDa MWCO. LCP mix was prepared with monoolein (Sigma) at a 1:1.5 protein to lipid ratio. A Mosquito LCP (TTP Labtech) was used to dispense a volume of 80 nl of LCP mixture onto a 96-well glass plate, which was covered with 750 nl of precipitant solution and sealed with a glass cover slip. Initial crystals appeared within 3 days, and were grown to optimal conditions up to 1 month in the following conditions: 100 mM NaCl, 100 mM Sodium Citrate, pH 6.1, 40 mM MgCl₂, 29% PEG 400, 500 uM L-myo-inositol-1-phosphate (prepared in house at ITQB from glucose by coupling hexokinase from Thermoproteus tenax and L-myo-inositol-1-phosphate synthase from Archaeoglobus fulgidus as described previously [12 (link)]), 2.5 mM CDP (Sigma) (MkPIPS-S79 bound to CDP and IP).

Cystinosin and nanobody P10 were incubated at a 1:1.5 molar ratio for 30 min and purified by SEC. Peak fractions containing cystinosin-P10 complex were concentrated to a final concentration of 60 mg/ml. Cystinosin-P10 complex was reconstituted into lipidic cubic phase (LCP) by mixing protein with monoolein (Sigma) at a 2:3 ratio (w/w) (Caffrey and Cherezov, 2009 (link)). Crystals were grown in 12% PEG400, 80 mM Li₂SO₄, and 50 mM MES pH 6.0 at 20 °C. Diffraction data were collected from multiple crystals, processed with HKL2000 (Otwinowski and Minor, 1997 (link)), and merged using XDS (Kabsch, 2010 (link)). The initial molecular replacement was carried out using truncated and modified nanobody Nb80 (Rasmussen et al., 2011 (link)) and truncated and modified EcSemiSWEET (Lee et al., 2015 (link)) as the search models in Phaser (McCoy et al., 2007 (link)). For the complex with Se-Met nanobody, selenium atoms of Se-Met nanobody were located by MR-SAD in Phaser. Model building and refinement cycles were carried out in Coot (Emsley and Cowtan, 2004 (link)) and Phenix (Adams et al., 2010 (link)). Structural figures were prepared using PyMOL (Schrödinger, LLC).

Inhibitor bound hENT1_cryst was crystallized with the lipidic cubic phase method⁵⁵ . Purified inhibitor-bound protein was concentrated to 40 mg ml⁻¹ prior to mixing with monoolein (Sigma) in a 40:60 weight-weight ratio. Crystallization was facilitated at room temperature in 96 well glass sandwich plates (MiTeGen) with 150 nL mesophase and 1.0 μL crystallization solution. The crystallization condition yielding the best diffracting crystals of the dilazep bound hENT1_cryst consisted of 35–50% PEG 400, 0.1M glycine pH 9.0 and 0.5 M NaCl. Plate-like crystals appeared 12 hours after setting up trays, and reached a full size of 50×20 μm in 3–5 days. Wells were opened with a tungsten-carbide glass cutter (MiTeGen), and crystals were harvested with 75 um MiTeGen micromounts and directly cooled in LN₂. Dilazep-transporter crystals were used for the heavy-atom soaks for phasing, and soaks were carried out as described previously⁵⁶ . For all heavy-atom screens, soaks were carried out for 24 hours prior to well re-opening and harvesting. For the NBMPR bound hENT1_cryst structure, the crystal was obtained from 30% PEG 500 MME, 0.1M magnesium chloride hexahydrate and 0.1M Tris-HCl pH 8.0. The crystal grew to full size after 7 days.