Letter | Published:

Near-atomic-resolution cryo-EM analysis of the Salmonella T3S injectisome basal body

Nature volume 540, pages 597601 (22 December 2016) | Download Citation


The type III secretion (T3S) injectisome is a specialized protein nanomachine that is critical for the pathogenicity of many Gram-negative bacteria, including purveyors of plague, typhoid fever, whooping cough, sexually transmitted infections and major nosocomial infections. This syringe-shaped 3.5-MDa macromolecular assembly spans both bacterial membranes and that of the infected host cell. The internal channel formed by the injectisome allows for the direct delivery of partially unfolded virulence effectors into the host cytoplasm1. The structural foundation of the injectisome is the basal body, a molecular lock-nut structure composed predominantly of three proteins that form highly oligomerized concentric rings spanning the inner and outer membranes2,3,4,5. Here we present the structure of the prototypical Salmonella enterica serovar Typhimurium pathogenicity island 1 basal body, determined using single-particle cryo-electron microscopy, with the inner-membrane-ring and outer-membrane-ring oligomers defined at 4.3 Å and 3.6 Å resolution, respectively. This work presents the first, to our knowledge, high-resolution structural characterization of the major components of the basal body in the assembled state, including that of the widespread class of outer-membrane portals known as secretins.

Access optionsAccess options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.


Primary accessions

Electron Microscopy Data Bank


  1. 1.

    , & Structural overview of the bacterial injectisome. Curr. Opin. Microbiol. 14, 3–8 (2011)

  2. 2.

    et al. A refined model of the prototypical Salmonella SPI-1 T3SS basal body reveals the molecular basis for its assembly. PLoS Pathog. 9, e1003307 (2013)

  3. 3.

    et al. The modular structure of the inner-membrane ring component PrgK facilitates assembly of the type III secretion system basal body. Structure 23, 161–172 (2015)

  4. 4.

    et al. A conserved structural motif mediates formation of the periplasmic rings in the type III secretion system. Nat. Struct. Mol. Biol. 16, 468–476 (2009)

  5. 5.

    et al. Structural characterization of the molecular platform for type III secretion system assembly. Nature 435, 702–707 (2005)

  6. 6.

    , & Secretins: dynamic channels for protein transport across membranes. Trends Biochem. Sci. 36, 433–443 (2011)

  7. 7.

    et al. Topology and organization of the Salmonella Typhimurium type III secretion needle complex components. PLoS Pathog. 6, e1000824 (2010)

  8. 8.

    & Three-dimensional model of Salmonella’s needle complex at subnanometer resolution. Science 331, 1192–1195 (2011)

  9. 9.

    et al. Interactions of the transmembrane polymeric rings of the Salmonella enterica serovar Typhimurium type III secretion system. MBio 1, (2010)

  10. 10.

    , , & Crystal structure of the N-terminal domain of the secretin GspD from ETEC determined with the assistance of a nanobody. Structure 17, 255–265 (2009)

  11. 11.

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

  12. 12.

    , & Essential role of a sodium dodecyl sulfate-resistant protein IV multimer in assembly-export of filamentous phage. J. Bacteriol. 178, 1962–1970 (1996)

  13. 13.

    , & Insertion of an outer membrane protein in Escherichia coli requires a chaperone-like protein. EMBO J. 15, 978–988 (1996)

  14. 14.

    et al. In vitro multimerization and membrane insertion of bacterial outer membrane secretin PulD. J. Mol. Biol. 382, 13–23 (2008)

  15. 15.

    , , & Multimerization-defective variants of dodecameric secretin PulD. Res. Microbiol. 162, 180–190 (2011)

  16. 16.

    , & Decoding the roles of pilotins and accessory proteins in secretin escort services. FEMS Microbiol. Lett. 328, 1–12 (2012)

  17. 17.

    et al. Structural characterization of the type-III pilot-secretin complex from Shigella flexneri. Structure 16, 1544–1554 (2008)

  18. 18.

    , , , & Structural and functional insights into the pilotin-secretin complex of the type II secretion system. PLoS Pathog. 8, e1002531 (2012)

  19. 19.

    , , , & Lipids assist the membrane insertion of a BAM-independent outer membrane protein. Sci. Rep. 5, 15068 (2015)

  20. 20.

    et al. Visualization of the type III secretion sorting platform of Shigella flexneri. Proc. Natl Acad. Sci. USA 112, 1047–1052 (2015)

  21. 21.

    et al. Outer membrane targeting of Pseudomonas aeruginosa proteins shows variable dependence on the components of Bam and Lol machineries. MBio 2, (2011)

  22. 22.

    et al. Assembly of the secretion pores GspD, Wza and CsgG into bacterial outer membranes does not require the Omp85 proteins BamA or TamA. Mol. Microbiol. 97, 616–629 (2015)

  23. 23.

    et al. Independent domain assembly in a trapped folding intermediate of multimeric outer membrane secretins. Structure 22, 582–589 (2014)

  24. 24.

    , & Sequential steps in the assembly of the multimeric outer membrane secretin PulD. J. Biol. Chem. 288, 30700–30707 (2013)

  25. 25.

    , , & Structure of a bacterial type III secretion system in contact with a host membrane in situ. Nat. Commun. 6, 10114 (2015)

  26. 26.

    et al. Bacterial secretins form constitutively open pores akin to general porins. J. Bacteriol. 196, 121–128 (2014)

  27. 27.

    , , & Effects of proteins on thermotropic phase transitions of phospholipid membranes. Biochim. Biophys. Acta 401, 317–335 (1975)

  28. 28.

    et al. Structure and electrophysiological properties of the YscC secretin from the type III secretion system of Yersinia enterocolitica. J. Bacteriol. 186, 4645–4654 (2004)

  29. 29.

    et al. Identification of the gate regions in the primary structure of the secretin pIV. Mol. Microbiol. 76, 133–150 (2010)

  30. 30.

    et al. Structural insights into the assembly of the type III secretion needle complex. Science 306, 1040–1042 (2004)

  31. 31.

    , , & Structure of the cholera toxin secretion channel in its closed state. Nat. Struct. Mol. Biol. 17, 1226–1232 (2010)

  32. 32.

    et al. Atomic model of the type III secretion system needle. Nature 486, 276–279 (2012)

  33. 33.

    et al. Architecture of the type IVa pilus machine. Science 351, aad2001 (2016)

  34. 34.

    & Contribution of Salmonella Typhimurium type III secretion components to needle complex formation. Proc. Natl Acad. Sci. USA 97, 11008–11013 (2000)

  35. 35.

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

  36. 36.

    & Measuring the optimal exposure for single particle cryo-EM using a 2.6Å reconstruction of rotavirus VP6. eLife 4, e06980 (2015)

  37. 37.

    & CTFFIND4: Fast and accurate defocus estimation from electron micrographs. J. Struct. Biol. 192, 216–221 (2015)

  38. 38.

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

  39. 39.

    RELION: implementation of a Bayesian approach to cryo-EM structure determination. J. Struct. Biol. 180, 519–530 (2012)

  40. 40.

    FREALIGN: high-resolution refinement of single particle structures. J. Struct. Biol. 157, 117–125 (2007)

  41. 41.

    , & Quantifying the local resolution of cryo-EM density maps. Nat. Methods 11, 63–65 (2014)

  42. 42.

    et al. High-resolution noise substitution to measure overfitting and validate resolution in 3D structure determination by single particle electron cryomicroscopy. Ultramicroscopy 135, 24–35 (2013)

  43. 43.

    et al. UCSF Chimera–a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612 (2004)

  44. 44.

    et al. Atomic-accuracy models from 4.5-Å cryo-electron microscopy data with density-guided iterative local refinement. Nat. Methods 12, 361–365 (2015)

  45. 45.

    , , & Features and development of Coot. Acta Crystallogr. D 66, 486–501 (2010)

  46. 46.

    et al. High-resolution comparative modeling with RosettaCM. Structure 21, 1735–1742 (2013)

  47. 47.

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

  48. 48.

    et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D 66, 12–21 (2010)

  49. 49.

    et al. EMRinger: side chain-directed model and map validation for 3D cryo-electron microscopy. Nat. Methods 12, 943–946 (2015)

  50. 50.

    , , , & ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010)

  51. 51.

    , , , & Electrostatics of nanosystems: application to microtubules and the ribosome. Proc. Natl Acad. Sci. USA 98, 10037–10041 (2001)

  52. 52.

    , & Quantitative comparison of caste differences in honeybee hemolymph. Mol. Cell. Proteomics 5, 2252–2262 (2006)

  53. 53.

    & MaxQuant enables high peptide identification rates, individualized p.p.b.-range mass accuracies and proteome-wide protein quantification. Nat. Biotechnol. 26, 1367–1372 (2008)

  54. 54.

    , & Improved allelic exchange vectors and their use to analyze 987P fimbria gene expression. Gene 207, 149–157 (1998)

  55. 55.

    & The Salmonella Typhimurium InvH protein is an outer membrane lipoprotein required for the proper localization of InvG. Mol. Microbiol. 28, 1367–1380 (1998)

  56. 56.

    et al. Three-dimensional reconstruction of the Shigella T3SS transmembrane regions reveals 12-fold symmetry and novel features throughout. Nat. Struct. Mol. Biol. 16, 477–485 (2009)

  57. 57.

    et al. Structure of the dodecameric Yersinia enterocolitica secretin YscC and its trypsin-resistant core. Structure 21, 2152–2161 (2013)

  58. 58.

    et al. Structural similarity of secretins from type II and type III secretion systems. Structure 22, 1348–1355 (2014)

  59. 59.

    et al. The PRoteomics IDEntifications (PRIDE) database and associated tools: status in 2013. Nucleic Acids Res. 41, D1063–D1069 (2013)

Download references


We thank A. Cheung for assistance with the expression trials of the PrgH130–392 GFP-fused basal body, C. Lizak for advice on GFP variants and membrane-protein purification and C. Yip and J. Rubenstein for advice on negative-stain TEM techniques. We thank UBC Bioimaging for access to TEM infrastructure. We thank K.-M. Moon and J. Rogalski at the Michael Smith Labs Proteomics Core Facility for assistance with LC–MS/MS. We thank F. Rosell at the LMB Spectroscopy and Kinetics Hub for assistance with circular dichroism. We thank S. Miller for providing the S. Typhimurium deletion strains and plasmids, as well as the InvG antibody. This work was funded by scholarships to L.W. and J.B. from the Canadian Institutes of Health Research (CIHR) and Michael Smith Foundation of Health Research, respectively, and operating grants from CIHR to N.C.J.S. and B.B.F., and the Howard Hughes International Senior Scholar program to N.C.J.S. N.C.J.S. is a Tier I Canada Research Chair in Antibiotic Discovery.

Author information

Author notes

    • L. J. Worrall
    •  & C. Hong

    These authors contributed equally to this work.

    • J. R. C. Bergeron
    •  & T. Spreter

    Present addresses: Department of Biochemistry, University of Washington, Seattle, Washington 98195, USA (J.R.C.B.); Zymeworks, Vancouver, British Columbia V6H 3V9, Canada (T.S.).


  1. Department of Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada

    • L. J. Worrall
    • , M. Vuckovic
    • , J. R. C. Bergeron
    • , D. D Majewski
    • , T. Spreter
    •  & N. C. J. Strynadka
  2. CryoEM Shared Resources, Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, Virginia 20147, USA

    • C. Hong
    • , R. K. Huang
    •  & Z. Yu
  3. Michael Smith Laboratories, University of British Columbia, Vancouver, British Columbia V6T 1Z4, Canada

    • W. Deng
    •  & B. B. Finlay


  1. Search for L. J. Worrall in:

  2. Search for C. Hong in:

  3. Search for M. Vuckovic in:

  4. Search for W. Deng in:

  5. Search for J. R. C. Bergeron in:

  6. Search for D. D Majewski in:

  7. Search for R. K. Huang in:

  8. Search for T. Spreter in:

  9. Search for B. B. Finlay in:

  10. Search for Z. Yu in:

  11. Search for N. C. J. Strynadka in:


L.J.W. performed all model building, refinement, structural analysis and modelling experiments. C.H. performed single particle cryo-EM grid preparation, data collection and map generation with input from R.K.H. and Z.Y. M.V. performed all cloning, basal body and secretin sample preparations used in the structure solution with input from L.J.W. and J.R.C.B., building on work initiated by T.S. L.J.W. and J.R.C.B. carried out negative-stain TEM analysis on basal body and secretin preps for quality control. L.J.W. and M.V. designed and made basal body mutants for secretion assays carried out by W.D. W.D. also generated/validated the invG deletion mutant. D.D.M. performed experiments probing isolated pilotin InvH and InvG S domain interactions. L.J.W. and N.C.J.S. principally wrote the manuscript with input from all.

Competing interests

The authors declare no competing financial interests.

Corresponding authors

Correspondence to Z. Yu or N. C. J. Strynadka.

Reviewer Information Nature thanks M. Beeby, A. Blocker, J. Rubinstein and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data

Supplementary information

PDF files

  1. 1.

    Supplementary Information

    This file contains original SDS-PAGE gels and anti-InvG western blots used to generate Fig. 3e (a, b), Extended Data Fig. 5f (a, b), Extended Data Fig. 8g (a, b), Extended Data Fig. 10c (c, d) and Extended Data Fig. 9a (e).

About this article

Publication history






Further reading


By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.