Fig. 3: Cryo-EM structural characterization of de novo-designed VHH binding to influenza haemagglutinin and TcdB.

a, Labelled cryo-EM two-dimensional class averages of designed VHH_flu_01 bound to influenza haemagglutinin (HA) strain A/USA:Iowa/1943 H1N1. b, The 3.0 Å cryo-EM three-dimensional reconstruction shows VHH_flu_01 bound to H1 along the stem in two protomers. c, Cryo-EM structure of VHH_flu_01 bound to influenza haemagglutinin. d, Superposition of the designed VHH CDR3 structure with the cryo-EM structure. e, Comparison of predicted CDR3 rotamers compared with the built 3.0 Å cryo-EM structure. f,g, The cryo-EM structure closely matches the design. h, Examination of apo haemagglutinin protomers juxtaposed with those bound to the designed VHH shows repositioning of glycan N296 to allow for binding of the designed VHH to the stem. i, Labelled cryo-EM two-dimensional class averages of the designed VHH, VHH_TcdB_H2, bound to full-length TcdB. j, The 4.6 Å cryo-EM three-dimensional reconstruction of the complex shows VHH_TcdB_H2 bound to the target epitope as predicted. CROPs, combined repetitive oligopeptides; GTD, glucosyltransferase domain. k, Owing to the modest resolution, a fragment of TcdB was first docked into the cryo-EM density map, and the full design model—including both the TcdB fragment and the designed VHH—was then aligned to the pre-fitted TcdB fragment. The predicted design closely matches the experimentally determined complex in structure, epitope targeting and overall conformation. l, Labelled cryo-EM two-dimensional class averages of the designed VHH, VHH_TcdB_H2_ortho, bound to full-length TcdB. m, The 5.7 Å cryo-EM three-dimensional reconstruction of the complex shows that VHH_TcdB_H2_ortho bound the target epitope as predicted. n, A TcdB fragment was docked into the cryo-EM map, followed by alignment of the full model including the OrthoRep-matured VHH. The resulting structure shows no detectable change in binding orientation or docking angle compared with the original design, indicating that OrthoRep maturation preserved the predicted mode of epitope engagement. In all panels: yellow indicates haemagglutinin; grey denotes the computational design prediction; pink or navy shows VHH (cryo-EM); and teal indicates glycan.