All dose-fractioned images were motion-corrected and dose-weighted by MotionCorr2(Zheng et al., 2017) software and their contrast transfer functions were estimated by ctffind4(Rohou and Grigorieff, 2015). A total of 2,039,214 particles were auto-picked using the model from SARS-CoV-2 Cap(−1)’-RTC (PDB: 7CYQ)(Yan et al., 2021) and extracted with a box size of 448 pixels in cryoSPARC(Punjani et al., 2017). The following 2D, 3D classifications and refinements were all performed in cryoSPARC. 887,588 particles were selected after two rounds of 2D classification based on complex integrity. This particle set was used to do Ab-Initio reconstruction in five classes, which were then used as 3D volume templates for heterogeneous refinement, with 135,801 particles converged into dCap(0)-RTC complex class and 80,256 particles converged into mCap(0)-RTC complex class. Next, these particles were imported into RELION 3.03(Scheres, 2012) to perform local classification to obtain one class particle with final resolution 3.35 Å and 3.78 Å, respectively. The methods are described in Figures S2 and andS4S4.

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