The bacterial type VI secretion system (T6SS) uses contraction of a long sheath to quickly thrust a tube with associated effectors across membranes of eukaryotic and bacterial cells1,2,3,4,5. Only limited structural information is available about the inherently unstable precontraction state of the T6SS. Here, we obtain a 3.7 Å resolution structure of a non-contractile sheath–tube complex using cryo-electron microscopy and show that it resembles the extended T6SS inside Vibrio cholerae cells. We build a pseudo-atomic model of the complete sheath–tube assembly, which provides a mechanistic understanding of coupling sheath contraction with pushing and rotating the inner tube for efficient target membrane penetration. Our data further show that sheath contraction exposes a buried recognition domain to specifically trigger the disassembly and recycling of the T6SS sheath by the cognate ATP-dependent unfoldase ClpV.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


  1. 1.

    Basler, M., Pilhofer, M., Henderson, G. P., Jensen, G. J. & Mekalanos, J. J. Type VI secretion requires a dynamic contractile phage tail-like structure. Nature 483, 182–186 (2012).

  2. 2.

    Hood, R. D. et al. A type VI secretion system of Pseudomonas aeruginosa targets a toxin to bacteria. Cell Host Microbe 7, 25–37 (2010).

  3. 3.

    MacIntyre, D. L., Miyata, S. T., Kitaoka, M. & Pukatzki, S. The Vibrio cholerae type VI secretion system displays antimicrobial properties. Proc. Natl Acad. Sci. USA 107, 19520–19524 (2010).

  4. 4.

    Mougous, J. D. et al. A virulence locus of Pseudomonas aeruginosa encodes a protein secretion apparatus. Science 312, 1526–1530 (2006).

  5. 5.

    Pukatzki, S. et al. Identification of a conserved bacterial protein secretion system in Vibrio cholerae using the Dictyostelium host model system. Proc. Natl Acad. Sci. USA 103, 1528–33 (2006).

  6. 6.

    Durand, E. et al. Biogenesis and structure of a type VI secretion membrane core complex. Nature 523, 555–560 (2015).

  7. 7.

    Leiman, P. G. et al. Type VI secretion apparatus and phage tail-associated protein complexes share a common evolutionary origin. Proc. Natl Acad. Sci. USA 106, 4154–4159 (2009).

  8. 8.

    Cianfanelli, F. R., Monlezun, L. & Coulthurst, S. J. Aim, load, fire: the type VI secretion system, a bacterial nanoweapon. Trends Microbiol. 24, 51–62 (2016).

  9. 9.

    Zoued, A. et al. Architecture and assembly of the type VI secretion system. Biochim. Biophys. Acta 1843, 1664–1673 (2014).

  10. 10.

    Basler, M. Type VI secretion system: secretion by a contractile nanomachine. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 370, (2015).

  11. 11.

    Brackmann, M., Nazarov, S., Wang, J. & Basler, M. Using Force to Punch Holes: Mechanics of Contractile Nanomachines. Trends Cell Biol. 623–632 (2017).

  12. 12.

    Ge, P. et al. Atomic structures of a bactericidal contractile nanotube in its pre- and postcontraction states. Nat. Struct. Mol. Biol. 22, 377–382 (2015).

  13. 13.

    Nakayama, K. et al. The R-type pyocin of Pseudomonas aeruginosa is related to P2 phage, and the F-type is related to lambda phage. Mol. Microbiol. 38, 213–231 (2000).

  14. 14.

    Kostyuchenko, V. A. et al. The tail structure of bacteriophage T4 and its mechanism of contraction. Nat. Struct. Mol. Biol. 12, 810–813 (2005).

  15. 15.

    Leiman, P. G. & Shneider, M. M. Contractile tail machines of bacteriophages. Adv. Exp. Med. Biol 726, 93–114 (2012).

  16. 16.

    Leiman, P. G., Chipman, P. R., Kostyuchenko, V. A., Mesyanzhinov, V. V. & Rossmann, M. G. Three-dimensional rearrangement of proteins in the tail of bacteriophage T4 on infection of its host. Cell 118, 419–429 (2004).

  17. 17.

    Heymann, J. B. et al. Three-dimensional structure of the toxin-delivery particle antifeeding prophage of Serratia entomophila. J. Biol. Chem. 288, 25276–25284 (2013).

  18. 18.

    Shikuma, N. J. et al. Marine tubeworm metamorphosis induced by arrays of bacterial phage tail-like structures. Science 343, 529–533 (2014).

  19. 19.

    Aksyuk, A. A. et al. The tail sheath structure of bacteriophage T4: a molecular machine for infecting bacteria. EMBO J. 28, 821–829 (2009).

  20. 20.

    Aksyuk, A. A. et al. Structural conservation of the myoviridae phage tail sheath protein fold. Structure 19, 1885–1894 (2011).

  21. 21.

    Clemens, D. L., Ge, P., Lee, B.-Y., Horwitz, M. A. & Zhou, Z. H. Atomic structure of T6SS reveals interlaced array essential to function. Cell 160, 940–951 (2015).

  22. 22.

    Kudryashev, M. et al. Structure of the type VI secretion system contractile sheath. Cell 160, 952–962 (2015).

  23. 23.

    Kube, S. et al. Structure of the VipA/B type VI secretion complex suggests a contraction-state-specific recycling mechanism. Cell Rep. 8, 20–30 (2014).

  24. 24.

    Basler, M. & Mekalanos, J. J. Type 6 secretion dynamics within and between bacterial cells. Science 337, 815 (2012).

  25. 25.

    Bönemann, G., Pietrosiuk, A., Diemand, A., Zentgraf, H. & Mogk, A. Remodelling of VipA/VipB tubules by ClpV-mediated threading is crucial for type VI protein secretion. EMBO J. 28, 315–325 (2009).

  26. 26.

    Förster, A. et al. Coevolution of the ATPase ClpV, the sheath proteins TssB and TssC, and the accessory protein TagJ/HsiE1 distinguishes type VI secretion classes. J. Biol. Chem. 289, 33032–33043 (2014).

  27. 27.

    Kapitein, N. et al. ClpV recycles VipA/VipB tubules and prevents non-productive tubule formation to ensure efficient type VI protein secretion. Mol. Microbiol. 87, 1013–1028 (2013).

  28. 28.

    Pietrosiuk, A. et al. Molecular basis for the unique role of the AAA+ chaperone ClpV in type VI protein secretion. J. Biol. Chem. 286, 30010–30021 (2011).

  29. 29.

    Vettiger, A., Winter, J., Lin, L. & Basler, M. The type VI secretion system sheath assembles at the end distal from the membrane anchor. Nat. Commun. 8, 16088 (2017).

  30. 30.

    Chang, Y.-W., Rettberg, L. A., Ortega, D. R. & Jensen, G. J. In vivo structures of an intact type VI secretion system revealed by electron cryotomography. EMBO Rep. 18, 1090–1099 (2017).

  31. 31.

    Brackmann, M., Wang, J. & Basler, M. VipA N-terminal linker and VipB-VipB interaction modulate the contraction of type VI secretion system sheath. Preprint at http://www.biorxiv.org/content/early/2017/06/21/152785 (2017).

  32. 32.

    Kimanius, D., Forsberg, B. O., Scheres, S. H. & Lindahl, E. Accelerated cryo-EM structure determination with parallelisation using GPUs in RELION-2. eLIFE 5, (2016).

  33. 33.

    Webb, B. & Sali, A. Comparative protein structure modeling using MODELLER. Curr. Protoc. Bioinformatics 54, 5.6.1–5.6.37 (2002).

  34. 34.

    Taylor, N. M. I. et al. Structure of the T4 baseplate and its function in triggering sheath contraction. Nature 533, 346–352 (2016).

  35. 35.

    Osipiuk, J. et al. Crystal structure of secretory protein Hcp3 from Pseudomonas aeruginosa. J. Struct. Funct. Genomics 12, 21–26 (2011).

  36. 36.

    Jobichen, C. et al. Structural basis for the secretion of EvpC: a key type VI secretion system protein from Edwardsiella tarda. PLoS ONE 5, e12910 (2010).

  37. 37.

    Ballister, E. R., Lai, A. H., Zuckermann, R. N., Cheng, Y. & Mougous, J. D. In vitro self-assembly of tailorable nanotubes from a simple protein building block. Proc. Natl Acad. Sci. USA 105, 3733–3738 (2008).

  38. 38.

    Brunet, Y. R., Henin, J., Celia, H. & Cascales, E. Type VI secretion and bacteriophage tail tubes share a common assembly pathway. EMBO Rep. 15, 315–321 (2014).

  39. 39.

    Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

  40. 40.

    Zoued, A. et al. Priming and polymerization of a bacterial contractile tail structure. Nature 531, 59–63 (2016).

  41. 41.

    Hagen, W. J. H., Wan, W. & Briggs, J. A. G. Implementation of a cryo-electron tomography tilt-scheme optimized for high resolution subtomogram averaging. J. Struct. Biol. 197, 191–198 (2016).

  42. 42.

    Mastronarde, D. N. Automated electron microscope tomography using robust prediction of specimen movements. J. Struct. Biol. 152, 36–51 (2005).

  43. 43.

    Kremer, J. R., Mastronarde, D. N. & McIntosh, J. R. Computer visualization of three-dimensional image data using IMOD. J. Struct. Biol. 116, 71–76 (1996).

  44. 44.

    Castaño-Díez, D., Kudryashev, M., Arheit, M. & Stahlberg, H. Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J. Struct. Biol. 178, 139–151 (2012).

  45. 45.

    Castaño-Díez, D., Kudryashev, M. & Stahlberg, H. Dynamo Catalogue: geometrical tools and data management for particle picking in subtomogram averaging of cryo-electron tomograms. J. Struct. Biol. 197, 135–144 (2017).

  46. 46.

    Li, X. et al. Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM. Nat. Methods 10, 584–590 (2013).

  47. 47.

    Frank, J. et al. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199 (1996).

  48. 48.

    Desfosses, A., Ciuffa, R., Gutsche, I. & Sachse, C. SPRING – an image processing package for single-particle based helical reconstruction from electron cryomicrographs. J. Struct. Biol. 185, 15–26 (2014).

  49. 49.

    Tang, G. et al. EMAN2: an extensible image processing suite for electron microscopy. J. Struct. Biol. 157, 38–46 (2007).

  50. 50.

    Rohou, A. & Grigorieff, N. CTFFIND4: fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015).

  51. 51.

    Diaz, R., Rice, W. J. & Stokes, D. L. in Methods in Enzymology (ed. Jensen, G. J.) 131–165 (Academic Press, Cambridge MA, 2010).

  52. 52.

    Egelman, E. H. The iterative helical real space reconstruction method: surmounting the problems posed by real polymers. J. Struct. Biol. 157, 83–94 (2007).

  53. 53.

    Kucukelbir, A., Sigworth, F. J. & Tagare, H. D. Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014).

  54. 54.

    Wang, R. Y.-R. et al. De novo protein structure determination from near-atomic resolution cryo-EM maps. Nat. Methods 12, 335–338 (2015).

  55. 55.

    Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of COOT. Acta Crystallogr. D 66, 486–501 (2010).

  56. 56.

    Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D 66, (213–221 (2010).

  57. 57.

    Cole, C., Barber, J. D. & Barton, G. J. The Jpred 3 secondary structure prediction server. Nucleic Acids Res 36, W197–W201 (2008).

  58. 58.

    Goddard, T. D., Huang, C. C. & Ferrin, T. E. Visualizing density maps with UCSF Chimera. J. Struct. Biol. 157, 281–287 (2007).

Download references


The work was supported by Swiss National Science Foundation (SNSF) grant 31003A_159525 and the University of Basel. H.S. acknowledges support from the SNSF NCCR TransCure. Calculations were performed at sciCORE (http://scicore.unibas.ch/) scientific computing core facility at the University of Basel. We acknowledge S. Ursich for the help in sample preparation for cryo-ET.

Author information


  1. Focal Area Infection Biology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056, Basel, Switzerland

    • Jing Wang
    • , Maximilian Brackmann
    •  & Marek Basler
  2. Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland

    • Daniel Castaño-Díez
    • , Kenneth N. Goldie
    •  & Henning Stahlberg
  3. BioEM Lab, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland

    • Daniel Castaño-Díez
  4. Max Planck Institute of Biophysics, Max-von-Laue Str. 3, 60438, Frankfurt am Main, Germany

    • Mikhail Kudryashev
  5. Buchmann Institute for Molecular Life Sciences, Max-von-Laue Str. 17, 60438, Frankfurt am Main, Germany

    • Mikhail Kudryashev
  6. Focal Area Structural Biology, Biozentrum, University of Basel, Klingelbergstrasse 50/70, CH-4056, Basel, Switzerland

    • Timm Maier
    •  & Henning Stahlberg


  1. Search for Jing Wang in:

  2. Search for Maximilian Brackmann in:

  3. Search for Daniel Castaño-Díez in:

  4. Search for Mikhail Kudryashev in:

  5. Search for Kenneth N. Goldie in:

  6. Search for Timm Maier in:

  7. Search for Henning Stahlberg in:

  8. Search for Marek Basler in:


J.W. collected cryo-electron microscopy data, performed image processing and generated atomic models. M.Br. isolated and purified the sheaths. M.K. performed some initial electron microscopy data collection and data analysis. D.C.-D. provided support and contributed to data analysis. K.N.G. and H.S. provided support and supervised data collection. T.M. contributed to and advised on atomic model building. M.Ba. conceived the project and analysed the data. M.Br., J.W. and M.Ba. wrote the manuscript. All authors read the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Marek Basler.

Electronic supplementary material

  1. Life Sciences Reporting Summary

    Supplementary Figures 1–5, Supplementary Tables 1–6, Supplementary Video legends.

  2. Life sciences reporting summary.

  3. Supplementary Video 1

    Details of tomography reconstruction of the wild-type extended sheath, VipA-N3 sheath structure and Hcp tube.

  4. Supplementary Video 2

    Proposed mechanism of T6SS assembly and contraction.

About this article

Publication history






Further reading