Abstract
R-type pyocins are representatives of contractile ejection systems, a class of biological nanomachines that includes, among others, the bacterial type VI secretion system (T6SS) and contractile bacteriophage tails. We report atomic models of the Pseudomonas aeruginosa precontraction pyocin sheath and tube, and the postcontraction sheath, obtained by cryo-EM at 3.5-Å and 3.9-Å resolutions, respectively. The central channel of the tube is negatively charged, in contrast to the neutral and positive counterparts in T6SSs and phage tails. The sheath is interwoven by long N- and C-terminal extension arms emanating from each subunit, which create an extensive two-dimensional mesh that has the same connectivity in the extended and contracted state of the sheath. We propose that the contraction process draws energy from electrostatic and shape complementarities to insert the inner tube through bacterial cell membranes to eventually kill the bacteria.
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Acknowledgements
We thank D. Martin of AvidBiotics for discussion and support throughout this project and UCLA undergraduate student J. Chiou for assistance in data processing. This research was supported in part by the US National Institutes of Health (NIH) (AI046420/AI094386 and GM071940 to Z.H.Z.). P.G. was supported in part by an American Heart Association Western States Affiliates Postdoctoral Fellowship (13POST17340020). We acknowledge the use of instruments at the Electron Imaging Center for Nanomachines, supported by UCLA and by instrumentation grants from the NIH (1S10RR23057). Recharge fees for access to this facility for imaging the pyocin samples were partially defrayed by an award to Z.H.Z. from the UCLA Clinical and Translational Science Institute core voucher program.
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Z.H.Z., J.F.M., P.G., D.S. and P.G.L. designed the experiments. Z.H.Z. supervised the execution of the experiments. D.S. prepared the crude pyocin sample. X.Y. purified the same sample. P.G. performed cryo-EM, processed the images and built the atomic models. All authors interpreted the results. P.G. and P.G.L. drafted the manuscript. P.G., P.G.L., Z.H.Z. and J.F.M. edited the manuscript, and all authors reviewed the final manuscript.
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J.F.M. is a cofounder, equity holder and chair of the scientific advisory board of AvidBiotics Inc., a biotherapeutics company in San Francisco.
Integrated supplementary information
Supplementary Figure 1 Surface charge of the top (left) and bottom (right) side of the tube hexamer.
Supplementary Figure 2 Electron microscopy images of the postcontraction pyocin R2.
Electron microscopy images of the pyocin R2 embedded in uranyl acetate stain (a) or vitreous ice (b). Arrows point to postcontraction pyocins.
Supplementary Figure 3 Conformational change during contraction.
(a and b) Superposition of a pre- and a postcontraction sheath subunit showing extend of conformation change in different regions during contraction. (c-g) Surface charge distributions of sheath subunits in their precontraction (c and d) and postcontraction (e-g) states. For clarity, the C domain of sheath is removed in panel g.
Supplementary Figure 4 Key interactions of neighboring sheath subunits in the precontraction state.
There are six neighbors for each sheath subunit. They are colored differently in the top panel; the central subunit is colored magenta. Between the magenta subunit and its six neighbors are interfaces, of which three are unique. Key residues or interactions on these interfaces are tabulated below.
Supplementary Figure 5 Key interactions of neighboring sheath subunits in the postcontraction state.
There are ten neighbors for each sheath subunit. They are colored differently in the top panel; the central subunit is colored magenta. Between the magenta subunit and its ten neighbors are interfaces, of which five are unique. Key residues or interactions on these interfaces are tabulated below.
Supplementary Figure 6 Resolution assessment and validation of the pre- and postcontraction structures.
(left column) Fourier shell correlation curves between maps calculated from half datasets (dark red) and between the atomic model and the map (navy blue) and R-Free factors (orange) are plotted in the same graph versus resolution, for the pre- (a) and postcontraction (b) states, respectively. (right column) Ramachandran plots for the pre- (a) and postcontraction (b) state structures. Occupancy of different areas in each plot is listed at the bottom side of it.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–6 (PDF 869 kb)
Fly-by animation of the 3D montage model of the pyocin R2
Fly-by animation of the 3D montage model of the pyocin R2 in its entity rendered as shaded colour surface (coloured by cylindrical radius). (MP4 55682 kb)
Shaded surface view of the 3D cryo-EM density map of the precontraction trunk
Views matching Figure 1d-g are shown consecutively. (MP4 54777 kb)
Density map of the attachment helix of the sheath
The density map of the attachment helix of the sheath is shown as mesh superimposed with its atomic model (sticks). (MP4 10117 kb)
Density map of a β-sheet region
The density map of a β-sheet region of the tube is shown as mesh superimposed with its atomic (MP4 21869 kb)
Structure of the contracted sheath
Fly-by animation of the density map of the contracted sheath rendered as semi-transparent surface superimposed with its atomic model (ribbons). This scene is matching Figure 6b. (MP4 76409 kb)
Morphing between pre- and postcontraction states of the sheath
The morphing between pre- and post-contraction states of the sheath is shown in two orthogonal views. Two sequences are shown with different colour schemes: in the first sequence, the colour scheme is similar to that in Figure 3a; in the second, the subunits within the same disc are shown in the same colour. (MP4 39917 kb)
Zoom-in view of Video 8
Morphing between Figure 6c to Figure 6d. (MP4 2337 kb)
Possible way to preserve the augmented β-sheet of the sheath during contraction
Figure 3e is morphed into its post-contraction counterpart, shown in three views with 45° horizontal rotation in between. (MP4 8277 kb)
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Ge, P., Scholl, D., Leiman, P. et al. Atomic structures of a bactericidal contractile nanotube in its pre- and postcontraction states. Nat Struct Mol Biol 22, 377–382 (2015). https://doi.org/10.1038/nsmb.2995
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DOI: https://doi.org/10.1038/nsmb.2995
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