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

Three microliters of the purified template and Remdesivir bound complex sample at about 15mg/ml (combined with 0.5 µL of 0.025% DDM) or the purified apo complex sample at about 0.75mg/ml (combined with 0.5 µL of 0.025% DDM) was applied onto a glow-discharged holey carbon grid (Quantifoil R1.2/1.3), blotted, and subsequently sample-coated grids were vitrified by plunging into liquid ethane using a Vitrobot Mark IV (Thermo Fischer Scientific). Cryo-EM imaging was performed on a Titan Krios equipped with a Gatan K2 Summit direct electron detector in the Center of Cryo-Electron Microscopy, Zhejiang University (Hangzhou, China). The microscope was operated at 300kV accelerating voltage, at a nominal magnification of 29,000× in counting mode, corresponding to a pixel size of 1.014 Å. 2,886 movies of the template and Remdesivir bound complex and 7,408 movies of the apo complex were obtained at a dose rate of about 8 electrons per Å 2 per second with a defocus ranging from -0.8 to -3.0 μm. The total exposure time was set to 8s with intermediate frames recorded every 0.2s, resulting in an accumulated dose of 64 electrons per Å2 and a total of 40 frames per micrograph. The dosefractionated image stacks were subjected to beam-induced motion correction using MotionCor2.1 (36) . A sum of all frames filtered according to the exposure dose in each image stack was used for further processing.