Crystal structure of Middle East respiratory syndrome coronavirus helicase

Hao, Wei; Wojdyla, Justyna Aleksandra; Zhao, Rong; Han, Ruiyun; Das, Rajat; Zlatev, Ivan; Manoharan, Muthiah; Wang, Meitian; Cui, Sheng

The reaction mixture (10μl) contained 50mM HEPES (pH 7.5), 5mM MgCl 2 , 2mM dithiothreitol (DTT), 1mM nucleotide (ATP, GTP, CTP or TTP), 50nM of partial duplex RNA substrate and 300nM unlabeled trap RNA (5'-CGAAGCUGCUAACAUCAG-3') [36] . The RNA substrate with 5' overhang is prepared by mixing a top strand: 5'-UUUUUUUUUUCUGAU GUUAGCAGCUUCG-3' with a bottom strand: 5'-HEX-CGAAGCUGCUAACAUCAG-3'. The RNA substrate with 3' overhang is prepared by mixing a top strand: 5'-CUGAUGUUAG CAGCUUCGUUUUUUUUUUUUUUUUUUUU-3' with the same bottom strand as in RNA partial duplex with 5' overhang. The reaction was initiated by the addition of MERS-CoV nsp13 (100nM) and incubated at 30˚C for 30 minutes. The reaction was terminated by addition of 2.5μl loading buffer (5X) containing 100 mM Tris-HCl (pH7.5), 1% SDS, 50mM EDTA and 50% glycerol. Samples were resolved by 10% native-PAGE running on ice. The gel was scanned with Typhoon Trio Variable Mode Imager (GE healthcare).