Fig. 1: PilQ forms the slime nozzle in filamentous cyanobacteria.

A–C Thin sections of high-pressure frozen Athrospira platensis cells show the tilted transpeptidoglycan channels harboring the nozzle apparatus (black arrows), which are arranged in a circumferential ring at each cross wall. B, C High magnification micrographs from the indicated regions in (A). To best visualize more channels, B shows an image selected from the thin section series of the area shown in (A). The black arrows indicate the position of the transpeptidoglycan channels that in cross sections are visible as small white dots (B) and in longitudinal sections as slightly angled less dark-stained bands traversing the peptidoglycan at an angle of 30-40° relative to the septum (C). Shown are representative images of a cell filament from two separate experiments, each involving over a hundred thin-sectioned filaments. D Platinum-carbon (Pt/C) shadowing of an isolated outer membrane patch reveals the rows of nozzles (black arrow) consisting of the peripheral ring (16–18 nm) and a central pore (6-8 nm) which have identical dimensions as the top views of the isolated pores (black arrows) in (F). The data presented are from a single experiment and have been validated by additional findings from negative staining (F). E Transmission electron microscope image of a negatively stained isolated nozzle preparation. Black arrows indicate individual double ring nozzles, while white arrows indicate linear arrays containing multiple nozzles. The length of an individual double nozzle is ca. 32 nm. F As has been observed for nozzles of other filamentous cyanobacteria, adsorption to grids without glow discharge reveals top views of the complex (black arrows), while only a few side views are visible (white arrows) demonstrating that the cyanobacterial and myxobacterial nozzles (Fig. 3C) share similar architecture. The image in (E) represents data from at least 200 independent isolation experiments, whereas F shows a representative image from five independent experiments. G Fractions from a slime nozzle enrichment were screened by TEM and scored for how many nozzles were observed per grid square (ND, not determined). Fractions were separated by SDS-PAGE, and two bands correlated with fractions enriched for nozzles, identified as PilQ (black arrow, higher MW band), and the pentapeptide repeat protein NIES39_A07680 (black arrow, lower MW band). Size markers (first lane) indicate molecular weight in kDa. The image shows a representative SDS-PAGE gel from ten independent experiments. H Integrative cryo-EM and AlphaFold 3D reconstruction of the isolated PilQ nozzle complexes. The ribbon diagrams on the left show AlphaFold’s structure predictions for PilQ (upper drawing) and NIES39_A07680 (lower drawing). The color bar below represents the per-residue % confidence metrics (predicted local distance difference test, pLDDT) of the two structures, with blue indicating high confidence. The PilQ reconstruction is segmented into domains but lacks the N0 domain (amino acid residues 1-240), which could not be matched with the electron density. The middle and right structures present various views of the integrative cryo-EM reconstruction and AlphaFold prediction of PilQ: A vertical cross-section through the PilQ-only reconstruction highlights a single PilQ monomer in red (middle top). The 3D reconstruction depicts the 16mer PilQ ring-shaped oligomer with attached NIES39_A07680 monomers in blue (middle bottom) and has a resolution of approximately 4 Å (Fig. S5). Lastly, a 3D reconstruction of the isolated double particles is shown, which strikingly resembles the nozzles previously isolated from Ph. uncinatum (see Fig. S7⁵³). Scale bars: A 1 μm; B 250 nm; C–F 200 nm. Source data are provided as a Source Data file.