Fig 1.
Cellular arrangement and structure of the P. aeruginosa PAO1 T4P.
a Slice through a tomogram of a P. aeruginosa PAO1 ΔpilT cell with T4Ps protruding from the cell. Cell membrane, T4Ps, as well as T4P secretion machinery, which assembles the T4Ps, are marked by arrows. b Cryo-EM micrograph of T4Ps purified from P. aeruginosa PAO1 ΔpilT cells, with T4Ps labelled by white arrows. The inset shows a representative 2D class average (scale bar 50 Å). c Cryo-EM density map of the assembled T4P reconstructed in RELION 4.0 with one of the PilA subunits highlighted in turquoise. The map resolution of 3.2 Å shows a clear separation of the β-strands in each of the PilA subunits (marked by red asterisks). TheT4P has a helical rise of 10.17 Å, a helical twist of 87.39° and thus a helical pitch of 41.89 Å.
Fig 2.
Structural features of the P. aeruginosa PAO1 T4P.
a Model of the assembled P. aeruginosa PAO1 T4P. b Cross section through the model showing the tightly packed N-terminal α-helices in the centre of the pilus, which in turn interact with helices in the globular domain of other pilin subunits (marked by red arrows). c Structure of an individual PilA subunit composing the pilus. The PilA subunit features an N-terminal α-helix and a globular domain consisting of an α-helical segment connected to a β-sheet by a linker called αβ-loop. At the C-terminus, an additional loop, called D-region, can be identified. The two helices (N-terminal and globular domain α-helices) are coupled by a ‘melted’ linker region. N- and C-termini are marked N and C, respectively. The inset highlights the strong hydrophobic interactions between the α-helix and β-sheet in the globular domain, which stabilise the pilin structure. d Hydrophobic subunit-subunit interactions in the core of the T4P are mediated by the PilA α-helices.
Fig 3.
Interactions between pilin subunits at the periphery of the pilus.
a Structural model of four neighbouring pilin subunits (labelled N, N+1, N-3, N-4 based on the helical arrangement). b Interaction between the N and the N-4 subunits mediated through loops resolved in our 3.2 Å-resolution cryo-EM map. c Interactions at the interface among subunits N, N-3 and N-4 via side chains resolved in our cryo-EM map. d Interactions at the interface between subunits N, N-3 and N+1, via side chains resolved in our cryo-EM map.
Fig 4.
Comparison of Type IV pilin structures from different organisms.
a-g Aligned structures of Type IV pilins from different organisms: P. aeruginosa PAO1 (this study, PDB: 9EWX) (a), E. coli (PDB: 6GV9) (b), N. gonorrhoeae (PDB: 5VXX) (c), T. thermophilus (PDB: 6XXD) (d), G. sulfurreducens (PDB: 6VK9) (e), M. xanthus (PDB: 8TJ2) (f), P. arsenaticum (PDB: 6W8U) (g). The pilins are coloured by domain: N-terminal helix (orange), melted segment (magenta), globular domain (blue). The pilin structures have been aligned to the P. aeruginosa pilin using the ChimeraX matchmaker function. The resulting orientations show the structural diversity between the pilins introduced by the presence of the melted linker region featured by most pilin structures. h P. aeruginosa PilA pilin ribbon diagram coloured based on structure conservation score from highly conserved (9) to low conservation (1) calculated by comparing the pilin structures presented in a-g by sequence alignment using ConSurf [87–89]. While the N-terminal α-helix is highly conserved, the globular domain of the pilin exhibits low conservation.
Fig 5.
Comparison of T4P architectures in different organisms.
a-g Models of T4P structures from different organisms with one pilin subunit highlighted in orange for each structure: P. aeruginosa PAO1 (this study, PDB: 9EWX) (a), E. coli (PDB: 6GV9) (b), N. gonorrhoeae (PDB: 5VXX) (c), T. thermophilus (PDB: 6XXD) (d), G. sulfurreducens (PDB: 6VK9) (e), M. xanthus (PDB: 8TJ2) (f), P. arsenaticum (PDB: 6W8U) (g). Flexible loops on the pilus surface are marked by red arrows.