Extended Data Fig. 4: Sequence alignment and lattice contact sites.

a, Sequence alignment of S. cerevisiae Pil1 and Lsp1 (MUSCLE) with domain architecture illustrated (dotted lines indicate unstructured regions in native-source MCC–eisosome model). Violet squares indicate sterol-binding residues, dodger squares indicate PS-binding residues, green squares indicate PI(4,5)P₂-binding residues, grey squares indicate other lipid-binding-pocket residues, red circles indicate residues that form lattice contacts. b–e, Lattice contact sites between Pil1 dimers. Previous nanometre-resolution helical reconstructions of reconstituted Pil1 and Lsp1 proteins revealed a lattice pattern that could be fitted with the Lsp1 crystal structure, albeit with unaccounted density at the lattice contact sites³³. Three regions of contact between the central dimer (red/salmon) and its neighbours (blue/light violet) are clear in our structures (b). The first site of contact is a short stretch of interactions between the well-folded, domain-swapped N-terminus (Nt) of monomer A (res1-8) in the central dimer (red) and the equivalent Nt stretch (res1-8) of monomer B in neighbouring dimer 2 (light violet) (c), including residue S6 which previously shown to be phosphorylated by Pkh1 and important for eisosome assembly in combination with other phosphorylated residues^78,79. The remaining two contact sites are localized to the BAR-domain tips, previously shown to be flexible in crystallographic studies³⁸. A stretch of electrostatic interactions between residues 171–186 on helix 3, as well as residue 145 on helix 2, of the BAR domain in monomer A of the central dimer (salmon) and the equivalent residues from monomer B of dimer 3 (blue) forms the second contact site (d). A hydrophobic interaction between Y155 at the tip of BAR-domain helix 2 on monomer A of the central dimer (salmon) with Y158 on monomer B of dimer 4 (light violet), and vice versa, forms a third contact (e).