Fig. 1: Tetrameric conformations of recombinant NA proteins vary across influenza strains and subtypes.

a NA structure and phylogeny. (Left) Model of NA structure as observed crystallographically, featuring a bound NA inhibitor (Zanamivir, green) and Ca²⁺ ions (orange). Model from PDB ID 4B7Q with the Ca²⁺ ion near the four-fold axis placed using an alignment with PDB ID 3NSS. (Right) Phylogenetic tree of NA of influenza A and B viruses. Scale bar, 0.1 amino acid substitutions per site. b Construct diagram for recombinant NA proteins used in this work. The globular head domain of NA (residues 83–469, N1 numbering) was genetically fused to the hVASP tetramerization domain (TD) by a flexible GG linker and a thrombin protease cleavage site. All constructs contained an N-terminal hexahistidine tag, and some constructs additionally contained a Strep Tag. See Supplementary Data 1 for amino acid sequences. c NS-EM 2D class averages of NA (scale bar, 10 nm). (Left) NA preparations from strains that show predominantly open or poorly ordered tetramers. (Middle) NA preparations from strains that show mixtures of closed and open tetramers. (Right) NA preparations from strains that show predominantly closed tetramers similar to those classically observed in NA crystal structures. d Cryo-EM 2D class averages of recombinant N1-CA09-WT (scale bar, 10 nm). e NS-EM 2D class averages of recombinant N1-CA09-WT and N1-MI15-WT maintained in the presence of 1 mM CaCl₂ throughout purification (scale bar, 10 nm).