Structural Basis for the Inhibition of the RNA-Dependent RNA Polymerase from SARS-CoV-2 by Remdesivir

Wanchao Yin; Chunyou Mao; Xiaodong Luan; Dan-Dan Shen; Qingya Shen; Haixia Su; Xiaoxi Wang; Fulai Zhou; Wenfeng Zhao; Minqi Gao; Shenghai Chang; Yuan-Chao Xie; Guanghui Tian; He-Wei Jiang; Sheng-Ce Tao; Jingshan Shen; Yi Jiang; Hualiang Jiang; Yechun Xu; Shuyang Zhang; Yan Zhang; H. Eric Xu

The eluted co-expressed RdRp complex was incubated with additional nsp7 and nsp8 from the bacterial expression in a 1:1:2 molar ratio and incubated at 4 °C for 4h. Incubated RdRp complex protein was concentrated with a 100 kDa molecular weight cut-off Centrifugal Filter (Millipore Corporation) and then size-separated by a Superdex S200 10/300 GL column in 25 mM HEPES pH 7.4, 300 mM sodium chloride, 0.1 mM magnesium chloride, 1 mM TCEP. The fractions for the monomeric complex were collected and concentrated for EM experiments of the apo complex. For the Remdesivir bound complex, the concentrated RdRp complex were diluted to 0.5 mg/ml with buffer of 25 mM HEPES pH 7.4, 100 mM sodium chloride, 2 mM magnesium chloride, 1 mM TCEP, combined with synthesized template-primer RNA and RTP in a 1:2:10 molar ratio and incubated at 4 °C for 2h. Then the incubated Remdesivir bound RdRp complex were concentrated for EM experiments.