High throughput crystallization screening kits

Initial crystallization screens for all 4 protein complexes were carried out at 20°C with a Gryphon crystallization robot (Art Robbins Instruments) using high-throughput crystallization screening kits (Hampton Research and Qiagen). Extensive manual optimizations were then performed at 20°C using the hanging-drop vapor-diffusion method when proteins were mixed with reservoir solution at 1:1 ratio.

Initial crystallization screens were carried out using a Gryphon crystallization robot (Art Robbins Instruments) with high-throughput crystallization screening kits (Hampton Research and Qiagen). Extensive manual optimization was then performed at 20°C using the hanging-drop vapor-diffusion method when proteins were mixed with reservoir solutions in 1:1 ratio. The H_CA–bSV2C complex was initially crystallized in a condition containing 100 mM sodium cacodylate, pH 6.5, 13% polyethylene glycol (PEG) 3,350, and 200 mM NaCl. The best crystals were obtained in the presence of 0.7% (v/v) 1-butanol, which was identified using an additive screen kit (Hampton Research). The crystals were cryo-protected in the original mother liquor supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen. The H_CA–gSV2C complex was originally crystallized as thin plates in a condition composed of 100 mM sodium acetate, pH 4.6, 20% PEG 3,350, and 200 mM ammonium phosphate monobasic. These crystals diffracted poorly. After extensive additive screening and optimization, the best crystals were obtained in the presence of 4% (w/v) polypropylene glycol P 400. The crystals were cryo-protected in the reservoir solution supplemented with 20% (v/v) ethylene glycol and flash-frozen in liquid nitrogen.