2.2. Bio-layer interferometry assay
The binding affinity between the various trimeric S glycoproteins and dimeric ACE2 receptors was evaluated using the Octet RED96 instrument at 30°C with a shaking speed of 1000 RPM (ForteBio), as described previously37. Anti-human IgG Fc biosensors (ForteBio) were used. Following 10 minutes of pre-hydration of anti-human IgG Fc biosensors and 1 minute of sensor check, 7.5 nM of hACE2-Fc or 15 nM of mvACE2-Fc in 10X kinetic buffer (ForteBio) were loaded onto the surface of anti-human IgG Fc biosensors for 5 minutes. After 1.5 minutes of baseline equilibration, 5 minutes of association was conducted at 10 – 100 nM S glycoprotein, followed by 5 minutes of dissociation in the same buffer used for baseline equilibration. For binding assays using mvACE2-Fc, the association was conducted with 25 – 200 nM S glycoprotein. The data were corrected by subtracting the signal from the reference sample, and a 1:1 binding model with the global fit was used to determine equilibrium dissociation constants (Kd). Statistical analysis was performed using GraphPad Prism 9 software. Two-way analysis of variance (ANOVA) with Dunnett’s multiple comparisons test was used to compare the binding affinity (Kd) of S glycoprotein variants with hACE2-Fc or mvACE2-Fc glycoproteins, and significance was defined as P <0.05.
2.3. Electron microscopy specimen preparation and data acquisition
To prepare cryo-EM samples, 3 µL of ~1.5 mg/mL mink S glycoprotein-mvACE2 complex (1:2.2 molar ratio S to mvACE2) were applied to glow-discharged holey carbon grids (Quantifoil Cu R1.2/1.3). The grids were blotted for 3 seconds at 100% relative humidity and flash-frozen in liquid ethane using a Vitrobot Mark IV system (Thermo Fisher Scientific). The cryo grids were imaged using a Krios cryogenic transmission electron microscope operated at 300 kV. The microscope was equipped with a Gatan K3 camera with a slit width of 20 eV, and a total of 11,392 micrographs were collected at 81,000X magnification. The image was exposed for 4.0 seconds fractionated with 40 frames with an accumulated dose of 49.98 e-2. The pixel size was 1.11 Å with a defocus range of -0.75 to -1.75 μm.
2.4. Cryo-EM data processing
All data processing was conducted in CryoSPARC v4.1.138. Patch motion correction and CTF estimation were performed before manually curating exposures to exclude 1,746 micrographs with statistical outliers. 4,458,674 particles were initially extracted via blob particle picking from the remaining 9,646 micrographs, followed by template particle picking. Extracted particles underwent a series of 2D classifications and 3D classifications for refinement. Local refinement was further performed with a mask covering the American mink S glycoprotein RBD interacting with mvACE2. The full workflow is summarized in Figure S1.