Em gp2 plunge freezer

Purified SARS-CoV-2 spike preparations were diluted to a concentration of ~1 mg/mL in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3. 2.5 μL of protein was deposited on a CF-1.2/1.3 grid that had been glow discharged for 30 seconds in a PELCO easiGlow™ Glow Discharge Cleaning System. After a 30 s incubation in >95% humidity, excess protein was blotted away for 2.5 seconds before being plunge frozen into liquid ethane using a Leica EM GP2 plunge freezer (Leica Microsystems). Frozen grids were imaged in a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan). Data were acquired using the Leginon system^{41 (link)}. The dose was fractionated over 50 raw frames and collected at 50ms framerate. This dataset was energy-filtered with a slit width of 30 eV. Individual frames were aligned and dose-weighted. CTF estimation, particle picking, 2D classifications, ab initio model generation, heterogeneous refinements, and homogeneous 3D refinements were carried out in cryoSPARC^{42 (link)}.

Purified Envelope SOSIP ectodomain preparations were prepared at concentrations of 4–5 mg/ml in 15 mM HEPES buffer with 150 mM NaCl at pH 7.1 and mixed with Fab at a 1:5 molar ratio. A total of 2.5 μl of the complex was deposited on a CF-1.2/1.3 grid that had been glow-discharged for 15 s in a PELCO easiGlow glow discharge cleaning system. After 30-s incubation in >95% humidity, the excess protein was blotted away for 2.5 s before being plunge-frozen in liquid ethane using a Leica EM GP2 plunge freezer (Leica Microsystems). Frozen grids were imaged in a Talos Arctica (Thermo Fisher) equipped with a K3 detector (Gatan). Individual frames were aligned, dose-weighted, and CTF corrected followed by particle picking, 2D classification, ab initio model generation, heterogeneous refinements, homogeneous 3D refinements, and local resolution calculations in cryoSPARC (Supp. Table 1) (113 ).

Purified SARS-CoV-2 spike preparations were diluted to a concentration of ~1 mg/mL in 2 mM Tris pH 8.0, 200 mM NaCl and 0.02% NaN3. 2.5 μL of protein was deposited on a CF-1.2/1.3 grid that had been glow discharged for 30 seconds in a PELCO easiGlow™ Glow Discharge Cleaning System. After a 30 s incubation in >95% humidity, excess protein was blotted away for 2.5 seconds before being plunge frozen into liquid ethane using a Leica EM GP2 plunge freezer (Leica Microsystems). Frozen grids for the N234A mutant were imaged in a Titan Krios (Thermo Fisher) equipped with a K3 detector (Gatan) while those for the N165A mutant were image in a Titan Krios equipped with a Falcon 3EC detector. Data were acquired using the Leginon system^{23 (link)} (N234A) or the EPU software (Thermo Fisher Scientific; N165A). The dose was fractionated over 50 raw frames and collected at 50ms framerate. This dataset was energy-filtered with a slit width of 30 eV for the N234A mutant. Individual frames were aligned and dose-weighted^{24 (link)}. CTF estimation, particle picking, 2D classifications, ab initio model generation, heterogeneous refinements, homogeneous 3D refinements and local resolution calculations were carried out in cryoSPARC^{25 (link)}.