4.5. Crystallization

All purified proteins were immediately crystallized at 18 °C. Initial screening was performed by using the sitting drop method on 96-Well MRC 2-Drop Crystallization Plates in polystyrene (SWISSCI, Neuheim, Switzerland) containing 90 µL of crystallization solution in the reservoir. 0.6 µL sample with 0.6 µL crystallization solution were spotted. For all three GlnKs, the protein sample was crystallized with and without 2 mM ATP, 2 mM 2OG and 2 mM MgCl₂. Since MtGlnK₁ and MtGlnK₂ with ligands aggregated strongly at 18 °C, the protein sample was first heated to 70 °C and centrifuged at 13,000× g for 5 min at room temperature to remove remaining aggregates before spotting.

MtGlnK₁ at a concentration of 33 mg/mL co-crystallized with ligands generated few crystals but none of them diffracted to an exploitable resolution. However, hexagonal rods instantly appeared for MtGlnK₁ at 33 mg/mL without ligands in 35% w/v Pentaerythritol propoxylate (17/8 PO/OH), 100 mM MES; pH 6.5 and 200 mM ammonium sulfate.

MtGlnK₂ at a concentration of 30 mg/mL without ligand crystallized as small squares in 35% w/v Pentaerythritol ethoxylate (15/4 EO/OH) and 100 mM HEPES pH 7.5. When MtGlnK₂ at 30 mg/mL was crystallized with ligands, high-quality shard shaped crystals appeared after minutes in 25% w/v Pentaerythritol ethoxylate (3/4 EO/OH) and 100 mM MES pH 6.5.

As stated above, four pools of purified MjGlnK₁ were obtained after gel filtration. The best crystals were obtained from the pool containing the protein eluted at 66 mL on the gel filtration. This pool, concentrated to 11.2 mg/mL, was co-crystallized with ligands and high-quality cube-shaped crystals appeared overnight in 20% w/v PEG 3350, 100 mM Bis-Tris propane pH 8.5 and 200 mM sodium nitrate.