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-/Å2. 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.