Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Structural characterization of the molecular platform for type III secretion system assembly


Type III secretion systems (TTSSs) are multi-protein macromolecular ‘machines’ that have a central function in the virulence of many Gram-negative pathogens by directly mediating the secretion and translocation of bacterial proteins (termed effectors) into the cytoplasm of eukaryotic cells1. Most of the 20 unique structural components constituting this secretion apparatus are highly conserved among animal and plant pathogens and are also evolutionarily related to proteins in the flagellar-specific export system. Recent electron microscopy experiments have revealed the gross ‘needle-shaped’ morphology of the TTSS2,3,4, yet a detailed understanding of the structural characteristics and organization of these protein components within the bacterial membranes is lacking. Here we report the 1.8-Å crystal structure of EscJ from enteropathogenic Escherichia coli (EPEC), a member of the YscJ/PrgK family whose oligomerization represents one of the earliest events in TTSS assembly5. Crystal packing analysis and molecular modelling indicate that EscJ could form a large 24-subunit ‘ring’ superstructure with extensive grooves, ridges and electrostatic features. Electron microscopy, labelling and mass spectrometry studies on the orthologous Salmonella typhimurium PrgK within the context of the assembled TTSS support the stoichiometry, membrane association and surface accessibility of the modelled ring. We propose that the YscJ/PrgK protein family functions as an essential molecular platform for TTSS assembly.

Your institute does not have access to this article

Relevant articles

Open Access articles citing this article.

Access options

Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

Figure 1: Stoichiometric analysis of S. typhimurium NC base components.
Figure 2: EscJ structure and intermolecular interactions.
Figure 3: EscJ forms a superhelix in the crystal but is anchored to the inner membrane in vivo.
Figure 4: Modelling and surface electrostatic analysis of the EscJ ring.
Figure 5: Surface mapping of S. typhimurium NC with limited biotinylation and MALDI–TOF MA.


  1. Ghosh, P. Process of protein transport by the type III secretion system. Microbiol. Mol. Biol. Rev. 68, 771–795 (2004)

    CAS  Article  Google Scholar 

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

    ADS  CAS  Article  Google Scholar 

  3. Kubori, T. et al. Supramolecular structure of the Salmonella typhimurium type III protein secretion system. Science 280, 602–605 (1998)

    ADS  CAS  Article  Google Scholar 

  4. Blocker, A. et al. Structure and composition of the Shigella flexneri ‘needle complex’, a part of its type III secreton. Mol. Microbiol. 39, 652–663 (2001)

    CAS  Article  Google Scholar 

  5. Kimbrough, T. G. & Miller, S. I. Assembly of the type III secretion needle complex of Salmonella typhimurium . Microbes Infect. 4, 75–82 (2002)

    CAS  Article  Google Scholar 

  6. Kimbrough, T. G. & Miller, S. I. Contribution of Salmonella typhimurium type III secretion components to needle complex formation. Proc. Natl Acad. Sci. USA 97, 11008–11013 (2000)

    ADS  CAS  Article  Google Scholar 

  7. Galan, J. E. & Collmer, A. Type III secretion machines: bacterial devices for protein delivery into host cells. Science 284, 1322–1328 (1999)

    ADS  CAS  Article  Google Scholar 

  8. Sukhan, A., Kubori, T., Wilson, J. & Galan, J. E. Genetic analysis of assembly of the Salmonella enterica serovar Typhimurium type III secretion-associated needle complex. J. Bacteriol. 183, 1159–1167 (2001)

    CAS  Article  Google Scholar 

  9. Crago, A. M. & Koronakis, V. Salmonella InvG forms a ring-like multimer that requires the InvH lipoprotein for outer membrane localization. Mol. Microbiol. 30, 47–56 (1998)

    CAS  Article  Google Scholar 

  10. Aizawa, S. I. Flagellar assembly in Salmonella typhimurium . Mol. Microbiol. 19, 1–5 (1996)

    CAS  Article  Google Scholar 

  11. Burghout, P. 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)

    CAS  Article  Google Scholar 

  12. Linderoth, N. A., Simon, M. N. & Russel, M. The filamentous phage pIV multimer visualized by scanning transmission electron microscopy. Science 278, 1635–1638 (1997)

    ADS  CAS  Article  Google Scholar 

  13. Nouwen, N. et al. Secretin PulD: association with pilot PulS, structure, and ion-conducting channel formation. Proc. Natl Acad. Sci. USA 96, 8173–8177 (1999)

    ADS  CAS  Article  Google Scholar 

  14. Jones, C. J., Macnab, R. M., Okino, H. & Aizawa, S. Stoichiometric analysis of the flagellar hook–(basal-body) complex of Salmonella typhimurium . J. Mol. Biol. 212, 377–387 (1990)

    CAS  Article  Google Scholar 

  15. Sekiya, K. et al. Supermolecular structure of the enteropathogenic Escherichia coli type III secretion system and its direct interaction with the EspA-sheath-like structure. Proc. Natl Acad. Sci. USA 98, 11638–11643 (2001)

    ADS  CAS  Article  Google Scholar 

  16. Suzuki, H., Yonekura, K. & Namba, K. Structure of the rotor of the bacterial flagellar motor revealed by electron cryomicroscopy and single-particle image analysis. J. Mol. Biol. 337, 105–113 (2004)

    CAS  Article  Google Scholar 

  17. Thomas, J., Stafford, G. P. & Hughes, C. Docking of cytosolic chaperone-substrate complexes at the membrane ATPase during flagellar type III protein export. Proc. Natl Acad. Sci. USA 101, 3945–3950 (2004)

    ADS  CAS  Article  Google Scholar 

  18. Leslie, A.G.W. Recent changes to the MOSFLM package for processing film and image plate data. Joint CCP4 + ESF-EAMCB Newsl. Protein Crystallogr. no. 26 (1992).

  19. Evans, P. R. Data reduction. Proc. CCP4 Study Weekend on Data Collection and Processing 114–122, (1993)

    Google Scholar 

  20. Terwilliger, T. C. & Berendzen, J. Automated MAD and MIR structure solution. Acta Crystallogr. D 55, 849–861 (1999)

    CAS  Article  Google Scholar 

  21. Terwilliger, T. C. Maximum-likelihood density modification. Acta Crystallogr. D 56, 965–972 (2000)

    CAS  Article  Google Scholar 

  22. McRee, D. E. XtalView/Xfit—A versatile program for manipulating atomic coordinates and electron density. J. Struct. Biol. 125, 156–165 (1999)

    CAS  Article  Google Scholar 

  23. Brunger, A. T. et al. Crystallography & NMR system: A new software suite for macromolecular structure determination. Acta Crystallogr. D 54, 905–921 (1998)

    CAS  Article  Google Scholar 

  24. Murshudov, G. N. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D 53, 240–255 (1997)

    CAS  Article  Google Scholar 

  25. Kabsch, W. A solution for the best rotation to relate two sets of vectors. Acta Crystallogr. A 32, 922–923 (1976)

    ADS  Article  Google Scholar 

  26. Nicholls, A., Sharp, K. A. & Honig, B. Protein folding and association: insights from the interfacial and thermodynamic properties of hydrocarbons. Proteins 11, 281–296 (1991)

    CAS  Article  Google Scholar 

  27. Kraulis, P. J. MOLSCRIPT: A program to produce both detailed and schematic plots of protein structures. J. Appl. Crystallogr. 24, 946–950 (1991)

    Article  Google Scholar 

  28. Merritt, E. A. B. & Bacon, D. J. Raster3D: photorealistic Molecular Graphics. Methods Enzymol. 277, 505–524 (1997)

    CAS  Article  Google Scholar 

Download references


We thank A. L. Lovering, C. P. C. Chiu and P. I. Lario for discussions; H. Law, K. Hayakawa, Y. Luo and Y. Wu for involvement in the early stages of the project; and the staff at the Advanced Light Source beamline 8.2.1 for data collection time and assistance. C.K.Y. is supported by fellowships from the Natural Sciences and Engineering Research Council of Canada and the Michael Smith Foundation for Health Research. N.C.J.S. and B.B.F. thank the Howard Hughes Medical Institute International Scholar Program, Canadian Institutes of Health Research and the Canadian Bacterial Diseases Network for funding. Funding for this project also came from grants from the NIH to S.I.M.Author Contributions C.K.Y completed the structural determination, analysis and modelling of EscJ, M.V. assisted in purification and crystallization of EscJ, R.A.P. developed the EscJ purification procedure, and E.A.F. did the original cloning of EscJ under the supervision of N.C.J.S. T.G.K. and H.B.F. performed the EM, labelling, and mass spectrometry experiments on Salmonella NCs under the supervision of S.I.M, and N.A.T. performed the EscJ localization and complementation assays under the supervision of B.B.F.

Author information

Authors and Affiliations


Corresponding author

Correspondence to Natalie C. J. Strynadka.

Ethics declarations

Competing interests

Coordinates and observed structure factors have been deposited to the Protein Data Bank under accession code 1YJ7. Reprints and permissions information is available at The authors declare no competing financial interests.

Supplementary information

Supplementary Table S1-S3

Table 1. Data collection and refinement details. Crystallographic data collection, structure determination and refinement details; Table 2. Identification of lysine-biotinylated tryptic peptides of PrgK by MALDI-TOF mass spectrometry. List of all tryptic peptides and modified lysine residues from limited biotinylation-mass spectrometry analysis of PrgK; Table 3. Identification of lysine-biotinylated tryptic peptides of PrgH by MALDI-TOF mass spectrometry. List of all tryptic peptides and modified lysine residues from limited biotinylation-mass spectrometry analysis of PrgH. (DOC 69 kb)

Supplementary Figure S1

Structure-based sequence alignment of EscJ with members of YscJ/PrgK family and flagellar FliF. (PDF 50 kb)

Supplementary Figure S2

PrgK isolated from the needle complex is palmitoylated. Two SDS-PAGE images showing palmitoylation of PrgK. (PDF 304 kb)

Supplementary Figure S3

Effects of triple mutation (E62A/K63A/E64A) on the structure and function of EscJ. A gel from EPEC secretion assay together with two detailed structural figures showing the triple mutation (E62A/K63A/E64A) does not affect function and structure of EscJ. (PDF 2452 kb)

Supplementary Figure Legends (DOC 26 kb)

Supplementary Video S1

EscJ ring model. A movie file illustrating the surface rendered representation of the EscJ ring model. (MP4 2574 kb)

Rights and permissions

Reprints and Permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI:

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.


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing