Extended Data Fig. 1: Sample preparation, functional characterization and in vitro product synthesis.

a, Elution profile of Gel-filtration chromatography (Superose 6 Increase 10/300 GL) of FKS1 purified in detergent GDN. Green dashed box marks the fractions pooled for cryoEM analysis. b, SDS-PAGE (left) and western blot (right) analysis of the fractions of the monodisperse elution peak in (a). Representative gel and western-blot image among 3 replicates are shown. c, Elution profile of Gel-filtration chromatography (Superose 6 Increase 10/300 GL) of FKS1 S643 purified in detergent GDN. Green dashed box marks the fractions pooled for cryoEM analysis and product synthesis. d, SDS-PAGE analysis of the fractions of the monodisperse elution peak in (c). A representative gel among 3 replicates is shown. e, Elution profile of Gel-filtration chromatography (Superdex 200 Increase 10/300 GL) of Rho1. Green dashed box marks the fractions pooled for activity assay and product synthesis. f, SDS-PAGE analysis of the fractions of the monodisperse elution peak in (e). Two peaks in (e) shows similar three-band pattern (f), and all three bands were identified as Rho1 by Mass Spec analysis. A representative gel among 3 replicates is shown. g, Western blot analyses of wild-type FKS1 and different FKS1 variants purified in detergent CHAPS. A representative image among 3 replicates is shown. h, SDS-PAGE analysis of GDN-purified flag-tagged FKS1 variants, which were used for the assays of activity (i-j) and product synthesis (k). First lane, FKS1 WT; second lane, FKS1 S643P (the echinocandin-resistant mutant); third lane, purified FKS1 S643P is immunodepleted by anti-flag beads; fourth lane, FKS1 S643P/K1261A. A representative gel among 3 replicates is shown. For (b,d,f,g,h), their full scans are provided in Supplementary Fig. 1. i, In vitro activity of FKS1 variants purified in GDN. The activity was assayed by monitoring UDP generated (1-h reaction). Data are mean ± SEM, n = 3 independent experiments. j–k, In vitro product synthesis assay by FKS1 variants purified in GDN. The assay was performed either by staining the synthesized products with dye aniline blue (j; 12-h reaction) or by visualization of the products synthesized (k; 48-h reaction). These experiments were repeated three times with similar results. l, Donor specificity of product synthesis by GDN-purified FKS1 S643P. Water-insoluble polymers (indicated by white arrow) appear only in the presence of UDP-Glc instead of UDP-GlcNAc. This experiment was repeated three times with similar results. m, The effects of Mg²⁺ on the catalytic activity of GDN-purified FKS1 S643P. Activity was assayed in the presence of 1 mM EDTA or 200 μM Mg²⁺. Data are mean ± SEM, n = 3 independent experiments. Inset, product synthesis in the presence of 1 mM EDTA or 200 μM Mg²⁺. This experiment was repeated three times with similar results.