Fig. 5. Cryo-EM structure of 7A3 and 8A2 nanobodies with SARS-Cov-2 spike.

(A) The structure models based on the EM images show that 8A2 and 7A3 bind two distinct sites on the RBD. 8A2 directly interferes with the ACE3 binding to the RBD. 7A3 binds a distinct conserved site on the RBD regardless of its conformational mode. 7A3 partially overlaps the ACE2 binding loci when bridging two RBDs. This interaction does not interfere with 8A2 binding, which is only observed in the up conformation. The mutations on the RBD derived from alpha (E484K, S494P, N501Y), beta (K417N, E484K, N501Y), gamma (K417T, E484K, N501Y), and delta (K417N, L452R, and T478K) were indicated. Green spheres, glycans. (B) The unique 7A3 binding pattern. The tip of 7A3 (7A3_A) is deeply buried in the structure of the spike, where the V_HH nanobody engages Asp985/B, Glu988/B, Pro987B and Pro384/A in the S2 subunit of the spike protein outside the RBD region. (C) Nanobodies epitopes coverage as determined from the experimental structure using a 5A contact cutoff. Sequences of coronavirus RBD for Bat_RaTG13 (A0A6B9WHD3); Human BJ01 (Q6GYR1); Pangolin (A0A6G6A2Q2); SARS-CoV-2 (P0DTC2) and SARS-CoV-1 (P59594) are presented for comparison purpose. Degree of sequence identity is indicated by background pink hue. 7A3 contact is indicated in blue and 8A2 in orange. Arrows indicate mutation sites of concern (black) and key ACE2 contact residues (green). RBM is indicated by the dotted box. Sequence alignment and colorization was performed using Jalview version 2.11.1. Alignments were performed using ClustallO and colorization by Blosum62.