Pil1 and Lsp1 form filaments in vitro. (A) Pil1 and Lsp1 aggregate in vitro. SDS-PAGE of factions of a sedimentation velocity gradient analyzing recombinant Pil1 and Lsp1. Protein marker sizes are indicated on the right. (B) Recombinant Pil1 and Lsp1 form filaments visualized by negative staining and EM. Pil1 assembles into ringlike structures as well as thin and thick filaments. Lsp1 mostly forms thick filaments. Bar, 100 nm. (C) Cryo-EM and tomographic reconstructions of Lsp1 filaments have a distinct striation pattern. Bar, 50 nm. (D) Averaged tomographic top, mid, and bottom sections of a thick Lsp1 filament. (E) Surface rendering of the Lsp1 filament reconstruction. (F) Classification of Lsp1 segments reveal classes differing in diameter (left panels; the narrow class is shown on the top, whereas the wider class is shown on the bottom). Power spectra of both major classes are characteristic for filaments of helical symmetry (middle panels) and reveal differences in geometry, also visible in the resulting 3D maps (right panels). Bar, 10 nm.