3.2. Cryo-EM structural determination
To facilitate structure determination, we prepared a trimeric mink S
glycoprotein, harboring some of the mutations (Δ69-70, Y453F, D614G)
found in mink cluster 5, with six proline substitutions that are
reported to have increased protein yields and stability in the prefusion
state35. We mixed the mink S glycoprotein with
monomeric mvACE2 at a molar ratio of 1:2.2 and incubated it for 15
minutes before subjecting the complex to vitrification for EM analysis.
Cryo-EM analysis of the complex determined four distinctive species:
mink S glycoprotein alone (13.8%), mink S glycoprotein bound to one
mvACE2 with downRBD (16.0%) or upRBD (14.3%), and mink S glycoprotein
bound to two mvACE2 in the upRBD position (55.8%) (Fig. 2). After 3D
classification and refinement, apo-mink S glycoprotein was resolved at
3.60 Å, while three conformational states of the mink S
glycoprotein-mvACE2 complex were captured and determined at 3.83 Å, 3.75
Å, and 3.36 Å. The RBDs were flexible with respect to the horizontal
plane of the spike protein, as shown by the 2D and 3D classification
analysis, capturing four different RBD angles: 27.1°, 28.9°, 62.8°, and
69.2° (Fig. S1F and 2).
Cryo-EM structural analysis of the mink S glycoprotein shows that its
overall organization of the trimer is similar to other variants,
including Alpha, Delta, Omicron, and previously published mink S
glycoprotein (Fig. 3). Our captured apo-mink S glycoprotein was
determined to have three receptor binding domains in the down position.
The RBDs of the mink S glycoprotein structures are less well-resolved,
most likely due to its flexibility in the region compared to the rest of
the protein chain. However, it is clear that the overall architecture of
the different S variants is conserved, which is consistent with the
conserved high affinity for hACE2 receptor.