Figure 4. O-linked sugar modifications of the LAL14/1 pilus.

a, Extra densities on residues threonine 83, serine 85 and threonine 90 indicate post-translational modifications.

b, Atomic model of the LAL14/1 pilus, with side chain oxygen atoms of serine and threonine shown as red spheres.

c, Violin plot showing the distributions of the sum of serine and threonine percentage in bacterial pilins (100–200 amino acids long, N=52,248, colored in green) and Sulfolobus proteins (100–200 amino acids long, N=7,326, colored in purple). The red dashed line indicates the corresponding percentage in LAL14/1 pilin Sil_2606. The box indicates the interquartile range, and the central line in the box indicates the median of the data.

d, Projections of the 3D reconstruction under high threshold (left) and low threshold (right). The low threshold projection reveals a fuzzy coat of peripheral density.

e, STEM analysis of LAL14/1 pili. Within one representative micrograph from 150 recorded (left), the white arrowhead indicates tobacco mosaic virus (control) and the blue arrowhead indicates a LAL14/1 pilus. The mass per length measurement for each segment are shown. The distribution of all mass/length values for the pili (right) yields an average of ~ 3.5 kDa/Å.

f, LAL14/1 pilus samples on silver stained SDS-PAGE before and after deglycosylation using trifluoromethanesulfonic acid (TFMS). This was performed twice, and the same bands were seen each time. Yellow arrowheads indicate the bands which contain SiL_2603 and SiL_2606 detected by MS/MS analysis. The red arrowhead indicates a higher mass band that was shown by MS/MS analysis to not contain either SiL_2603 or Sil_2606 (but contained a membrane-associated ESCRT-III-2 protein) and thus could not be due to a more heavily glycosylated species of Sil_2603 or Sil_2606. This figure (f) is reproduced in Supp Fig. 9 for purposes of comparison.