Epu version 2

Purified spike protein was buffer exchanged into 2 mM Tris pH 8.0, 200 mM NaCl, 0.02% NaN₃ buffer using a desalting column (Zeba, Thermo Fisher). A final concentration of 0.2 mg/mL was incubated with CR3022 Fab (in the same buffer) in a 6:1 molar ratio (Fab to trimeric spike) at room temperature. Aliquots were taken at 50 min and 3 h and 3 μL immediately applied to a holey carbon-coated 200 mesh copper grid (C-Flat, CF-2/1, Protochips) that had been freshly glow discharged on high for 20 s (Plasma Cleaner PDC-002-CE, Harrick Plasma) and excess liquid removed by blotting for 6 s with a blotting force of −1 using vitrobot filter paper (grade 595, Ted Pella Inc.) at 4.5°C, 100% relative humidity. Blotted grids were then immediately plunge frozen using a Vitrobot Mark IV (Thermo Fisher).
Frozen grids were first screened on a Glacios microscope operating at 200 kV (Thermo Fisher) before imaging on a Titan Krios G2 (Thermo Fisher) at 300 kV. Movies (40 frames each) were collected in compressed tiff format on a K3 detector (Gatan) in super resolution counting mode using a custom EPU version 2.5 (Thermo Fisher) with a defocus range of 0.8-2.6 μm and at a nominal magnification of x105,000, corresponding to a calibrated pixel size of 0.83 Å/pixel, see Table S4.

Purified recombinant omicron spike and prototypic spike (4 µl at ~3.3 µM and ~10 µM, respectively), supplemented with 8 mM CHAPSO immediately before grid preparation, were applied to freshly glow-discharged Quantifoil R1.2/1.3 300-mesh copper grids (EM Sciences). Grids were blotted for 4 s at 22 °C under 100% chamber humidity and plunge-frozen in liquid ethane using a Vitrobot Mark IV (FEI). Cryo-EM data were collected using EPU version 2.5 (ThermoFisher Scientific) on a Titan Krios electron microscope (ThermoFisher Scientific) equipped with a Falcon III direct electron detector (ThermoFisher Scientific) at the Hormel Institute, University of Minnesota. The movies were collected at a nominal magnification of 96,000×, corresponding to 0.89 Å per pixel, at a dose rate of 0.95 e⁻ per pixel per second with a defocus ranging from −1.0 to −2.4 µm. Each micrograph consists of 32 dose-framed fractions and was recorded with a total dose of 40 e⁻/Ų. The statistics of cryo-EM data collection are summarized in Supplementary Table 2.