Extended Data Fig. 5: Predicted location of TMH1.

(a) Relationship of evolutionarily coupled residues within Cv-HAS’ TM and GT regions, generated in MapPred based on 65,535 sequences. TMH1 is shown at its predicted location as a violet cylinder. (b) RoseTTAfold models of full-length Cv-HAS. Cv-HAS is shown as a surface and its TMH 2 as a blue cylinder. TMH1 is shown as a cartoon at its predicted locations. (c) An AlphaFold2 predicted structure of human HAS2 (coloured blue to red from its N- to C-terminus) overlaid with the Cv-HAS structure shown as a grey cartoon and semi-transparent surface. (d) TMH1 remains disordered when two cytosolic nanobodies are used for cryo-EM analyses. (e) Catalytic activity of TMH1 truncated Cv-HAS. Left: Western blot of IMVs used for in vitro activity measurements. Right: Catalytic activity of the indicated Cv-HAS mutants expressed relative to the wild type enzyme. The assay quantifies ³H-labelled HA by scintillation counting. Control reactions in the absence of UDP-GlcA served as background and are subtracted. Error bars represent deviations from the means with n = 3 independent experiments.