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 purified RdRp complex is relatively thermostable with a melting temperature of 53°C ( Figure S1G ). Negative-stain EM visualization of the nsp12-nsp7-nsp8 complex displayed monodispersed particles in excellent homogeneity ( Figure S1H ). For the apo nsp12-nsp7-nsp8 complex, we vitrified the sample in the presence of the detergent DDM. The initial attempt of image processing reveals that the particles are preferentially oriented ( Figure S2 ). Therefore, we collected and processed over 7400 micrograph movies of more than 5.7 million particle projections to increase the projection amount from the non-preferential orientation, out of which 81,494 particles are used to yield a density map of 2.8 Å resolution. Cryo-EM studies of the nsp12-nsp7-nsp8 complex bound to the template-primer RNA and RTP (termed as the template-RTP RdRp complex) were first hampered by the two facts. First, most particles were adsorbed to cryo-EM grid bars rather than staying in the vitreous ice. Second, RNA duplex dissociated from the template-RTP RdRp complex likely owning to the adverse effects during cryo-EM specimen preparation. Eventually, we prepared the cryo-EM specimen of the template-RTP RdRp complex at the concentration of 15 mg/ml supplemented with DDM prior to vitrification ( Figure S3 ). We collected 2886 micrograph movies, which yielded a 2.5 Å resolution structure using 130,386 particle projections. Because of the relatively high resolution of our structures, EM density map is clear for key structure features across the complex ( Figure S4 ).