Protein crystallization

Gel filtration-purified proteins (20 mg each) were mixed with the TEV protease (1 mg ml⁻¹, 500 μl) individually and incubated overnight at 4 °C to cleave their polyhistidine tags. The mixture was then loaded onto a Ni-NTA column and the flow-through fraction was collected and concentrated to 2 ml before being purified by gel-filtration in the gel-filtration buffer. Each of the purified proteins was concentrated to 1.35 mM and used to screen for 800 conditions for crystallization. The seleno-methionine-labelled erWalK6R86M protein was crystallized in a buffer containing 0.2 M ammonium citrate dibasic, 20% w/v polyethylene glycol 3350 and pH 5.1 by sitting-drop method. The crystals were cryoprotected with a reservoir solution containing 22% glycerol and were frozen in liquid N₂.