Crystallization of the LACV Gc head domain

Initial attempts to produce two different constructs of the LACV Gc head domain were unsuccessful. However, the complete variable region (aa 477–911) could readily be produced in S2 cells. Following affinity purification, the protein was further purified by gel permeation chromatography in 20 mM Tris-Cl pH 8.0, 150 mM NaCl on a HiLoad Superdex 200 pg column (GE Healthcare). As no crystals could be obtained with this construct, a sample of 0.7 mg/mL was proteolytically trimmed with 2 µg/mL trypsin (Sigma-Aldrich) for 30 min at 24 °C. The reaction was stopped with 5 µg/mL soybean trypsin inhibitor (Sigma-Aldrich), and the largest cleavage product with an apparent molecular mass of ~30 kDa was again purified by gel permeation chromatography in 20 mM Tris-Cl pH 8.0, 150 mM NaCl. Optimal crystals of this fragment were obtained by the hanging-drop vapor diffusion method: 0.75 µL of a 11.1 mg/mL protein sample in 20 mM Tris-Cl pH 8.0, 150 mM NaCl were added to 0.75 µL of reservoir solution containing 0.1 M HEPES pH 7.5, 10% v/v 2-propanol, 20% w/v PEG 4 K. The drops were equilibrated against reservoir solution on siliconized glass slides (Hampton) for 5 days at 18 °C. Crystals were cryo-protected by immersion in 17% v/v glycerol, 83 mM HEPES pH 7.5, 8.3% v/v 2-propanol, 16.6% w/v PEG 4 K for less than 30 s prior to conservation in liquid nitrogen.