Cryo-EM Sample Preparation and Data Analysis

Samples for cryo-EM study were prepared essentially as described^{15 (link),52 (link)} (Supplementary Table 4). All EM grids were evacuated for 2 min and glow-discharged for 30 s using a plasma cleaner (Harrick PDC-32G-2). Four microliters of spike protein (0.8 mg ml⁻¹) was mixed with the same volume of Fabs (1 mg ml⁻¹ each), and the mixture was immediately applied to glow-discharged holy-carbon gold grids (Quantifoil, R1.2/1.3) in an FEI Vitrobot IV (4 °C and 100% humidity). Data collection was performed using either a Titan Krios G3 equipped with a K3 direct detection camera, or a Titan Krios G2 with a K2 camera, both operating at 300 kV. Data processing was carried out using cryoSPARC (v3.2.1)^{53 (link)}. After 2D classification, particles with good qualities were selected for global 3D reconstruction and then subjected to homogeneous refinement. To improve the density surrounding the RBD–Fab region, UCSF Chimera (v1.16)^{54 (link)} and Relion (v3.1)^{55 (link)} were used to generate the masks, and local refinement was then performed using cryoSPARC (v3.2.1). Coot (v0.8.9.2)^{56 (link)} and Phenix (v1.20)^{57 (link)} were used for structural modelling and refinement. Figures were prepared using USCF ChimeraX (v1.3)^{58 (link)} and Pymol (v2.4.0, Schrödinger, LLC.).