Cryo-EM movies of the samples were collected on a Titan Krios electron microscope (Thermo Fisher Scientific) operated at an accelerating voltage of 300 kV with a nominal magnification of ×22,500 (table S1). The movies were recorded on a K2 Summit direct electron detector (Gatan) operated in the super-resolution mode (yielding a pixel size of 1.02 Å after two times binning), under a low-dose condition in an automatic manner using SerialEM (50). Each frame was exposed for 0.15 s, and the total accumulation time was 6.45 s, leading to a total accumulated dose of 50 e2 on the specimen. To solve the problem of preferred orientation associated with SARS-CoV-2 S trimer, we additionally collected tilt datasets with the stage tilt at 30° or 40°, while the other conditions remained the same.

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