Figure 6.

Affinity driving mutations in Omicron RBD have previously been identified by in vitro evolution for tighter binding

(A) Analysis of the occurrence and prevalence of Omicron-variant mutations. The background is colored according to S-protein functional domains. The four positions critical for the high affinity of RBD-62 are highlighted in bold. Mutation frequencies within individual lineages are denoted in green (100%–75%), blue (75%–50%), and magenta (50%–25%). Information about the distribution and frequency of S-protein mutations and the spatiotemporal characterization of SARS-CoV-2 lineages were retrieved from www.outbreak.info (Mullen et al., 2020) and GISAID database (Elbe and Buckland-Merrett, 2017).^∗ Same evolutionary origin, ^anumber of evolutionary non-related lineages with given or similar mutation (Zahradnik et al., 2021c), ^blog(10) number of the observed Omicron mutation at the given position as determined on 14^th November 2021, ^csame as ^bbut total log(10) number of changes at the given position. ^dFold-change in binding as determined by yeast-surface display. Fold-change is the ratio between original RBD KD and the mutant RBD KD for binding human ACE2.

(B) Comparison of fold-change in binding affinity among selected mutations and their combinations as determined by titrating ACE2 on yeast surface displayed RBD mutations. For Omicron, yeast titration is denoted in violet, SPR (this study) is dark red, SPR as determined in Cameroni et al. (2021) is gray and ELISA as determined in Schubert et al. (2021) is in orange. Data denoted by black dots have been reported previously (Zahradník et al., 2021b).

(C) RBD-62 (blue)/ACE2 (green) structure (PDB: 7BH9) overlaid on Omicron RBD structure (orange) as determined bound to Beta-55. All Omicron mutations are shown, overlaid on relevant RBD-62 mutations.

(D) Electrostatic potential surface depictions calculated using PyMol. Blue is positive and red negative potential (scale bar shown below).