Fig. 3. Cryo-EM structure of the CTC-445.2–S complex.

(A to D) Cryo-EM reconstructions of CTC-445.2 (blue) bound to soluble spike trimers (gray). 3D classification revealed four distinct classes: one CTC-445.2 bound to an “up” RBD (A), two CTC-445.2 bound to two “up” RBDs (B), two CTC-445.2 bound to one “up” and one “down” RBD (C), and three CTC-445.2 bound to two “up” and one “down” RBD (D). (E) Overlay of CTC-445.2-RBD computationally modeled (yellow) and experimentally determined using cryo-EM (blue). The Cα RMSD between the design model and the refined experimental structure is 1.1 Å. (F to H) Comparison of cryo-EM CTC-445.2 (blue), computationally modeled CTC-445.2 (yellow), and hACE2 (green) at the interface of the RBD (gray). (I) Deep mutational scanning heatmap showing the average effect on the enrichment for single site mutants of CTC-445.2 when assayed by yeast display for binding to the SARS-CoV-2 RBD (binding assayed at RBD concentrations of 100, 50, 25, 12.5, 6.25, 3.125, and 1.5625 pM; see the materials and methods). (J) Design model of CTC-445.2 colored by average enrichment at each residue position [from the data in (I)] bound to SARS-CoV-2 RBD (gray). As expected, mutations in the core of the design or to positions involved in binding to the RBD are generally disallowed. The deep mutational scanning revealed that there is still room to further improve the binding affinity of CTC-445.2, including mutations in the binding interface that in principle could afford higher potency and selectivity at the cost of compromising the decoy’s mutational escape resiliency (see Fig. 4).