Fig. 3: Epitope determination for CR3022 and ACE2.

Epitope comparison between cryo-ER and alanine-scanning mutagenesis by mammalian cell surface display. The color for each residue corresponds to the normalized log mean binding per expression. Gray color represents the same binding as the negative control, red and blue represent decreased and increased binding, respectively. The brown residue R355 represents a structural site of RBD where a loss of binding occurred for all antibodies tested. A Binding effect of all mutated residues for CR3022 and ACE2. The key-residues were K378 and Y380 for CR3022 and Y473 for ACE2. B, C Visual representation of the CR3022 epitope. The green area represents residues of RBD in proximity of CR3022 as determined with cryo-ER. The residues F377, K378, Y380, and H519, located within the green area, were identified as the four most contributing residues for antibody binding. The three most prominent residues K378, Y380, and F377 made up a hot spot region where K378 and Y380 almost completely remove antibody binding when mutated to alanine. D, E Visual representation of the ACE2 epitope. The gray area are residues of RBD in proximity of ACE2 as determined using X-ray crystallography. Residues Y449, Y473, F486, and R466 were the top four contributing residues for ACE2 binding, with Y473 identified as the most prominent residue where mutation to alanine almost completely abolished ACE2 binding towards RBD.