For sample-specific details, refer to Table S6.

Movies were collected in compressed tiff format on a Titan Krios G2 (Thermo Fisher) operating at 300 kV with a K3 detector (Gatan) in super resolution counting mode using a custom version of EPU 2.5 (Thermo Fisher). A defocus range of 0.8-2.6 μm was applied with a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel and with a total dose of 43-47 e/ Å2, see Table S6.

Two-times binned movies were then motion corrected and aligned on the fly using Relion(3.1) scheduler (Zivanov et al., 2018) with a 5 × 5 patch based alignment. CTF-estimation of full-frame non-weighted micrographs was performed with the GCTF (1.06) (Zhang, 2016) module in cryoSPARC(v2.14.1-live) (Punjani et al., 2017).

Data for 88, 150, 158 were collected using a Titan Krios G2 (Thermo Fischer) operating at 300 kV with a K2 camera and a GIF Quantum energy filter (Gatan) with a 30 eV slit. For 159 (IgG), 384 and 316, data were collected as for 88, 150 and 158, except using a 20 keV slit. Rapid multi-shot data acquisition was set up using custom scripts with SerialEM (version 3.8.0 beta) (Mastronarde, 2005) at a nominal magnification of 165 kX, corresponding to a calibrated pixel size of 0.82 Å per pixel. A defocus range of −0.8 μm to −2.6 μm was used with a total dose of ~45-57 e-2 applied across 40 frames. Motion and CTF correction of raw movies was performed on the fly using cryoSPARC live patch-motion and patch-CTF correction (Punjani et al., 2017).

Poor-quality images were discarded after manual inspection of CTF and motion estimations. Particles were then blob picked in cryoSPARC (Punjani et al., 2017) and initially extracted with four times binning. After inspection of 2D classes, classes of interest were selected to generate templates for complete particle picking. Binned particles were then subjected to one to three rounds of reference free 2D classification followed by 3D classification with an ab-initio derived model before further refinement and unbinning.

For both 150 and 158, two data separate data collections were set up on the same grid, and refined particle sets from each collection were separated by exposure groups before being combined. For 150, a total of 77,265 exposure-group split particles were initially combined (51,554 from 4726 movies and 25,711 from 2079 movies), re-classified into five classes, and the two best classes (42,655 particles) subjected to further non-uniform refinement, with obvious density for Fab bound to one RBD in an ‘up’ conformation. Notably, discarded classes included a high proportion of undecorated S (28,463 particles, 4.4 Å reported resolution at GSFSC = 0.143, −43 Å2 B-factor).

Classification using heterogeneous refinement in cryoSPARC was found to be generally poor, and, instead, 3D variability analysis was employed to try to better resolve full spike-Fab structures. Local refinements were also performed with masks focused around the Fab/RBD region (not reported here), but maps were still insufficient to clearly build a model at the RBD/Fab interface and far inferior to the crystallographic maps. 3D variability analysis was found to be essential for isolating the RBD up and RBD down conformations for 159-IgG. Results from this are presented for 159-IgG and 384. Briefly, data were separated into eight clusters using the 3D variability analysis module with a 6 Å resolution filter and a mask around the RBD/Fab region. Masks were generated by initially rigid body fitting a model of the spike and a Fab into a refined map in Chimera before selecting an area of the model including the RBD and fab and using the ‘color zone’ module to crop out this desired part of the map. The resulting map was smoothed with a Gaussian filter (Pettersen et al., 2004), converted into a mask format using Relion3.1 ‘Mask Create’ before import into cryoSPARC. Resolution estimates quoted in the Table S6 were taken from Gold standard-FSC (FSC = 0.143) reported in the local resolution module in cryoSPARC (Punjani et al., 2017).

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