Fig. 2: Stabilization of the closed tetrameric state of N1 CA09 NA.

a Design and analysis pipeline for stabilization of closed tetrameric NAs using homology-directed mutations and computational protein design. Models constructed using PDB entries 4B7Q, 6BR5, and 1USE. Electron microscope image was generated by BioRender. b Structural depiction of the four different spaces in NA targeted for design. Two views of the CA09 NA tetramer are shown. c NS-EM 2D class averages (top) and designed mutations (bottom) in a series of CA09 NA mutants that exhibit varying degrees of tetramer closure (scale bar, 10 nm), and explore the roles of spaces A, B, and D in tetramer closure and expression (scale bar, 10 nm). 2D class averages for N1-CA09-WT are the same as presented in Fig. 1c. d Thermal denaturation of N1-CA09-WT and N1-CA09-sNAp-155 in the presence of 1 mM CaCl₂ (closed circles and solid lines) or 5 mM EDTA (open circles and dashed lines), monitored by intrinsic tryptophan fluorescence. Top panels show raw data, while lower panels show smoothed first derivatives used to calculate melting temperatures. The barycentric mean (BCM) of the fluorescence emission spectra is plotted. e SLS during thermal denaturation of N1-CA09-WT and N1-CA09-sNAp-155 in the presence of 1 mM CaCl₂ or 5 mM EDTA, with aggregation temperatures shown for CaCl₂-treated samples. Data are plotted as in panel d.