Index screen

Crystal screening was done at room temperature by the sitting-drop vapor diffusion method using 96-well plates (Greiner) and a range of commercial screens. A Mosquito nanolitre robot (TTP Labtech) was used to set up 200 nl sitting drops.
Crystals of untagged fibromodulin were obtained from a 10 mg/ml protein solution after one month at condition C6 of the Wizard Classic 3 screen (Rigaku) and at condition G5 of the Index screen (Hampton Research), but these crystals did not diffract to high resolution when cryoprotected with 30% glycerol. A range of crystallisation conditions and cryoprotectants were screened. The best results were obtained by growing the crystals at 26% (w/v) PEG 3350, 200 mM lithium sulphate monohydrate, 100 mM Tris-HCl (pH 8.5) and using an additional 15% PEG 3350 as cryoprotectant.
The chondroadherin-AB31 complex crystallised under a wide range of conditions at acidic pH, but the crystals were later found to contain only chondroadherin. When tested, chondroadherin alone crystallised under the same conditions. The best crystals grew from a 10 mg/ml protein solution in 20 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM CaCl₂ using 200 mM monobasic potassium phosphate (pH 4.8), 20% (w/v) polyethylene glycol 3350 as precipitant. Crystals were harvested in reservoir solution supplemented with 20% ethylene glycol and flash-frozen in liquid nitrogen.

NmGHR crystallization conditions were screened using protein concentrated up to 6.6 mg/mL in 50 mM Tris–HCl pH 7.5, 3.5 M NaCl. Best diffracting crystal was obtained from the Index Screen (Hampton Research) using the Phoenix crystallization robot (Art Robbins). Crystals appeared at 21°C in a droplet formed by mixing equal volumes (0.5 μL) of the protein solution, and reservoir solution, containing polyethylene glycol (PEG) 3350 25% w/v, 0.1 M Tris–HCl pH 8.5 and 0.2 M lithium sulfate. Crystal was cryoprotected by increasing the PEG 3350 concentration up to 30% w/v before flash-freezing. Data were collected at the ID23-2 beamline of ESRF synchrotron (Grenoble, Fr), at a wavelength of 0.873 Å. Data were processed using XDS (Kabsch, 2010 (link)), Pointless and Aimless (Winn et al., 2011 (link)). Final statistics are reported in Table 1.

Purified mitoNEET protein (20 mg/ml) mixed with an equal molar concentration of furosemide was screened by sitting drop vapor diffusion against Index screen (Hampton Research). Red crystals formed in under a week in several conditions. Final data collection was performed using crystals grown by sitting drop vapor diffusion by mixing a 1:1 ratio of protein with 60% v/v Tacsimate pH 7.0. The mother liquor was used for cryoprotection. Data collection was performed on the Northeastern Collaborative Access Team beamline 24-ID-C at the Advanced Photon Source at the iron peak. Data were indexed, integrated, and scaled using iMOSFLMand Aimless. To avoid model bias molecular replacement was combined with anomalous signals (MR-SAD) using Phaser and PDB 3REE as the search model^{15 (link)}. Refinement was performed using phenix²⁷ . Model building/rebuilding was performed using Coot^{28 (link)}. OMIT maps were constructed by removing the ligand from the final refined structure file, performing three macro-cycles of refinement with simulated annealing, and calculating a |F_o|—|F_c| map with phenix.maps.