Native crystal: Crystals were grown by in situ proteolysis with trypsin. Trypsin was added in a 1:200 (w/w) ration to 20 mg/mL native His6-TF-AbWSD1 before set up of the crystallization screens. 100 nL precipitant (17.3 % ethanol, 0.2 M MgCl2, 0.1 M TRIS pH 8.0) were added to 200 nL protein using 96-well plates with a Cartesian pipetting robot (Zinsser Analytic). Plates were stored at 277.15 K. Crystals were transferred into a cryoprotectant consisting of mother liquor supplemented with 30 % ethylene glycol and then flash cooled in liquid nitrogen.
277.15
2
蒸気拡散法, シッティングドロップ法
SeMet crystal: Trypsin was added in a 1:1000 ratio (w/w) to 15 mg/mL selenomethionine-labelled His6-TF-AbWSD1. Sitting drops consisting of 100 nL protein and 100 nL precipitant (14.1 % ethanol, 0.1 M MgCl2, 0.1 M HEPES pH 7.5) were pipetted with a Gryphon robot (Art Robbins Instruments). The 96-well plates were stored at 277.15 K. For cryoprotection of the crystals, 2-methyl-2,4-pentanediol was added to the crystallization drop by transferring it five times with a 0.4 mm LithoLoop (Molecular Dimensions) at 277.15 K. Crystals were then harvested from the drop and flash-cooled in liquid nitrogen.