REL 2024-04-11 2024-07-19 2024-11-19 0000-0001-7268-0306 Tao Ni School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong 21 Sassoon Road, Pokfulam Hong Kong China 00000 0000-0001-7268-0306 Tao Ni School of Biomedical Sciences, LKS Faculty of Medicine, The University of Hong Kong 21 Sassoon Road, Pokfulam Hong Kong China 0 Huang YX Ni T The University Grants Committee, Research Grants Council (RGC) #27103023 Hong Kong 10.3 10.6019/EMPIAR-12038 ssET organism Ni T Huang YX Zhong LJ Huang YX English 10.1016/s41586-024-07817-y

Sample: Purified SARS-CoV-2 DMV nsp3-4 pore complex Data collection: The tilt-series were acquired using Thermofisher Krios equipped with a Falcon 4i camera and Selectris energy filter, with a slit width of 20 eV. A dose-symmetric scheme (group of 2) was used, with a tilt range of -51° to 51° (or -60° to 60°) at 3° increments and an exposure dose of 3 e-/Å2 per image. The motion-corrected tilt-series and the corresponding IMOD transformation files, tilt angle which are required by RELION4 are uploaded.

Tilt-series of purified SARS-CoV-2 double membrane vesicles formed by nsp3-4 /data tilt series MRC MRC 4635 35 32 BIT FLOAT 4096 1.56 4096 1.56

The tilt-series were acquired using Thermofisher Krios equipped with a Falcon 4i camera and Selectris energy filter, with a slit width of 20 eV. A dose-symmetric scheme (group of 2) was used, with a tilt range of -51° to 51° (or -60° to 60°) at 3° increments and an exposure dose of 3 e-/Å2 per image, and 35 or 41 frames per image. The raw micrographs were motion-corrected in MRC format (5x5 patch using relion_motioncor) and then stacked into individual tilt-series. The tilt-series alignment results (including the transformation matrix *.xf, local transformation file and tilt angle) for RELION4 subtomogram averaging pipeline are provided. The particle_data.star contains the metadata of refined subtomograms. The pixel size of the tilt-series is 1.56 Å and then calibrated into 1.571 Å.