Fig 5. R1a-B6 key residues on Hemagglutinin A(H1N1)pdm09 crystal structure.

(A) A total of 5151 full-length HA sequences corresponding to H1N1, H5N1, H2N2 and H9N2 viral subtypes were aligned and the relative diversity at non-conserved positions was evaluated and showed as a logo sequence (S3 Table). Alignment of HA2 Gly¹²-Asn⁶⁰ is shown. Residues predicted to form part of the epitope footprint of our stem binding sdAb panel and identified by yeast display and deep sequencing indicated by black arrows (Fig 5B and 5C). Non-conserved residues either within subtype or across subtypes are highlighted by grey boxes. (B) Residues that show diversity but are buried in the HA structure, positioned on the reverse face of the HA monomer or at the interface of a HA trimer were not considered for mutagenesis and testing (black residues) (S3 Table). Residues that vary across viral subtypes are surface exposed and close in the structure to the binding footprint defined by deep sequencing and to residues tested in Fig 5 (residues highlighted in red) were chosen for experimental testing (orange residues).