Use of a combined cryo-EM and X-ray crystallography approach to reveal molecular details of bacterial pilus assembly by the chaperone/usher pathway

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Many bacteria assemble hair-like fibers termed pili or fimbriae on their cell surface. These fibers mediate adhesion to various surfaces, including host cells, and play crucial roles in pathogenesis. Pili are polymers composed of thousands of individual subunit proteins. Understanding how these subunit proteins cross the bacterial envelope and correctly assemble at the cell surface is important not only for basic biology but also for the development of novel antimicrobial agents. The chaperone/usher pilus biogenesis pathway is one of the best-understood protein secretion systems, thanks largely to innovative efforts in biophysical techniques such as X-ray crystallography and cryo-electron microscopy. Such a combined approach holds promise for further elucidating remaining questions regarding the multi-step and highly dynamic pilus assembly process, as well as for studying other protein secretion and organelle biogenesis systems.

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Introduction to the combined approach of X-ray crystallography and cryo-electron microscopy

Single particle cryo-electron microscopy (SPEM) is a biophysical method for visualizing the structures of macromolecular assemblies [1]. SPEM starts with purified protein complexes in solution. Individual molecules in solution at random orientation are pipetted onto an EM grid and rapidly frozen in liquid ethane to achieve buffer vitrification. Frozen samples are kept at low temperature during electron imaging in a transmission electron microscope. A large number of particle images are

References and recommended reading

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Acknowledgements

Work in the Li laboratory is supported by NIH grants AI70285 and GM74985 and LDRD grant 06-60 at the Brookhaven National Laboratory under contract with the US Department of Energy. Work in the Thanassi laboratory is supported by NIH grants GM62987 and AI55621.

References (51)

  • A. Holmgren et al.

    Crystal structure of chaperone protein PapD reveals an immunoglobulin fold

    Nature

    (1989)
  • M. Vetsch et al.

    Mechanism of fibre assembly through the chaperone-usher pathway

    EMBO Rep

    (2006)
  • D.C. Dodd et al.

    Kinetic analysis of the synthesis and assembly of type 1 fimbriae of Escherichia coli

    J Bacteriol

    (1984)
  • H. Remaut et al.

    Fiber formation across the bacterial outer membrane by the chaperone/usher pathway

    Cell

    (2008)
  • T.W. Ng et al.

    The usher N terminus is the initial targeting site for chaperone-subunit complexes and participates in subsequent pilus biogenesis events

    J Bacteriol

    (2004)
  • A.R. Osborne et al.

    Protein translocation is mediated by oligomers of the SecY complex with one SecY copy forming the channel

    Cell

    (2007)
  • J. Frank

    Three-Dimensional Electron Microscopy of Macromolecular Assemblies: Visualization of Biological Molecules in their Native State

    (2006)
  • K. Mitra et al.

    Ribosome dynamics: insights from atomic structure modeling into cryo-electron microscopy maps

    Annu Rev Biophys Biomol Struct

    (2006)
  • G.E. Murphy et al.

    In situ structure of the complete Treponema primitia flagellar motor

    Nature

    (2006)
  • T.C. Marlovits et al.

    Assembly of the inner rod determines needle length in the type III secretion injectisome

    Nature

    (2006)
  • T.C. Marlovits et al.

    Structural insights into the assembly of the type III secretion needle complex

    Science

    (2004)
  • C.H. Jones et al.

    FimH adhesin of type 1 pili is assembled into a fibrillar tip structure in the Enterobacteriaceae

    Proc Natl Acad Sci U S A

    (1995)
  • M.J. Kuehn et al.

    P pili in uropathogenic E. coli are composite fibres with distinct fibrillar adhesive tips

    Nature

    (1992)
  • D. Verger et al.

    Molecular mechanism of P pilus termination in uropathogenic Escherichia coli

    EMBO Rep

    (2006)
  • E.T. Saulino et al.

    Snapshots of usher-mediated protein secretion and ordered pilus assembly

    Proc Natl Acad Sci U S A

    (2000)
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