For crystallization, the GP was treated with Endo-β-acetylglucosaminidase F1 at 37 °C for 1 hour, further purified with size exclusion chromatography and concentrated to 10-12 mg/ml. Crystallization screen experiments were carried out using the nanolitre sitting-drop vapour diffusion method in 96-well plates as previously described35 (link). Crystals were initially obtained from Hampton Research PEGRx™ 1 screen, condition 37 containing 10% (w/v) PEG 6,000 and 0.1 M Sodium citrate tribasic dihydrate (pH 5.0). The best crystals were grown in optimized condition containing 9% (w/v) PEG 6,000 and 0.1 M Sodium citrate tribasic dihydrate at pH 5.2.
To obtain GP–inhibitor complexes, crystal soaking experiments were performed. Crystal soaking solutions were prepared by first dissolving the inhibitors in 100% DMSO and then diluting with 15% (w/v) PEG 6,000 and 0.1 M Sodium citrate tribasic dihydrate (pH 5.0) to a final DMSO concentration of 10%. The inhibitor concentration was typically from 1 to 10 mM depending on solubility. Crystals were soaked in the above solutions for between 20 minutes and 5 hours. Crystals soaked for longer normally diffracted less well and the crystals from which the GP-toremifene and GP-ibuprofen complex structures were obtained were only soaked for 20 minutes.
To obtain GP–inhibitor complexes, crystal soaking experiments were performed. Crystal soaking solutions were prepared by first dissolving the inhibitors in 100% DMSO and then diluting with 15% (w/v) PEG 6,000 and 0.1 M Sodium citrate tribasic dihydrate (pH 5.0) to a final DMSO concentration of 10%. The inhibitor concentration was typically from 1 to 10 mM depending on solubility. Crystals were soaked in the above solutions for between 20 minutes and 5 hours. Crystals soaked for longer normally diffracted less well and the crystals from which the GP-toremifene and GP-ibuprofen complex structures were obtained were only soaked for 20 minutes.