Chymotrypsinogen

We determined crystallization conditions for chymotrypsinogen (Sigma) using the commercial Crystal Screen HT (Hampton Research) set up with a Mosquito robot (STP Labtech). Crystals were grown via hanging-drop vapor diffusion at 18°C from condition F11. To optimize the crystallization conditions further, we set up a 24-well tray using hanging-drop vapor diffusion with a fixed pH of 6.5, varying the concentration of both dioxane (10% or 15%) and ammonium sulfate (1.0–2.0 M). Each drop, consisting of 1 µl reservoir solution (1.0–2.0 M ammonium sulfate, 0.1 M MES pH 6.5 and 10% or 15% dioxane) and 1 µl 10.0 mg ml⁻¹ enzyme, was equilibrated over 0.5 ml reservoir solution. The other two crystallization conditions consisted of either 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6, 30%(w/v) PEG 4000 or 0.5 M ammonium sulfate, 0.1 M sodium citrate tribasic dihydrate pH 5.6, with 1.0 M lithium sulfate monohydrate in the reservoir. All crystals were cryocooled in 3.5 M lithium sulfate. A typical drop containing these crystals is shown in Fig. 1 ▸.