Structural basis for the regulatory function of a complex zinc-binding domain in a replicative arterivirus helicase resembling a nonsense-mediated mRNA decay helicase

Deng, Zengqin; Lehmann, Kathleen C.; Li, Xiaorong; Feng, Chong; Wang, Guoqiang; Zhang, Qi; Qi, Xiaoxuan; Yu, Lin; Zhang, Xingliang; Feng, Wenhai; Wu, Wei; Gong, Peng; Tao, Ye; Posthuma, Clara C.; Snijder, Eric J.; Gorbalenya, Alexander E.; Chen, Zhongzhou

Purified nsp10Á was concentrated to 10 mg/ml and initial crystallization trials were performed at 16 C using the sitting-drop vapour-diffusion method by mixing 1 ml of protein solution with 1 ml of reservoir solution. The conditions were then optimized and high-quality crystals were obtained in 1.6 M (NH 4 ) 2 SO 4 , 0.1 M HEPES, pH 7.1, 25 mM KCl and 20% ethylene glycol. To obtain crystals of the protein-DNA complex, purified protein and partially double-stranded DNA with a 5 0 single-stranded poly-thymidine overhang (the two partially complementary sequences were 5 0 -TTTTTTTTTTGCAGTGCT CG-3 0 and 5 0 -CGCGAGCACTGC-3 0 ) were mixed in a 1:1.5 molar ratio and incubated at 4 C overnight. The complex was further purified by size-exclusion chromatography (Superdex 200, GE Healthcare) and concentrated to 5 mg/ml. The condition for obtaining crystals was 14% PEG 3350, 0.1 M HEPES, pH 7.0, and 0.2 M calcium acetate. For data collection, crystals were cryoprotected in mother liquor containing 25% (v/v) ethylene glycol and flash cooled to À173 C.