High-Resolution Structural Analysis of the Kinesin-Microtubule Complex by Electron Cryo-Microscopy

  • Keiko Hirose
  • Linda A. Amos
Part of the Methods in Molecular Biology™ book series (MIMB, volume 392)


To understand the interaction of kinesin and microtubules, it is necessary to study the three-dimensional (3D) structures of the kinesin-microtubule complex at a high enough resolution to identify structural components such as α-helices and β-sheets. Electron cryo-microscopy combined with computer image analysis is the most common method to study such complexes that cannot be crystallized. By selecting microtubules that have a helical symmetry, 3D structures of the complex can be calculated using the helical 3D reconstruction method. Details of the interaction are studied by docking the individual crystal structures of the kinesin motor domains and tubulin heterodimer into the 3D maps of the complex. To study the structural changes during ATP hydrolysis, structures of the complexes in the presence and absence of different nucleotides are compared.

Key Words

Kinesin microtubule molecular motors electron microscopy three-dimensional reconstruction 


  1. 1.
    Hirose, K., Lockhart, A., Cross, R.A., and Amos, L.A. (1995) Nucleotide-dependent angular change in kinesin motor domain bound to tubulin. Nature 376, 277–279.CrossRefPubMedGoogle Scholar
  2. 2.
    Sosa, H., Hoenger, A., and Milligan, R.A. (1997) Three different apporoaches for calculating the three-dimensional structure of microtubules decorated with kinesin motor domains. J. Struct. Biol. 118, 149–158.CrossRefPubMedGoogle Scholar
  3. 3.
    Hoenger, A., Sack, S., Thormahlen, M., Marx, A., Muller, J., Gross, H., and Mandelkow, E. (1998) Image reconstructions of microtubules decorated with monomeric and dimeric kinesins: comparison with x-ray structure and implications for motility. J. Cell Biol. 141, 419–430.CrossRefPubMedGoogle Scholar
  4. 4.
    Hirose, K., Cross, R.A., and Amos, L.A. (1998) Nucleotide-dependent structural changes in dimeric NCD molecules complexed to microtubules. J. Mol. Biol. 278, 389–400.CrossRefPubMedGoogle Scholar
  5. 5.
    Ray, S., Meyhofer, E., Milligan, R.A., and Howard, J. (1993) Kinesin follows the microtubule’s protofilament axis. J. Cell Biol. 121, 1083–1093.CrossRefPubMedGoogle Scholar
  6. 6.
    Meurer-Grob, P., Kasparian, J., and Wade, R.H. (2001) Microtubule structure at improved resolution. Biochemistry 40, 8000–8008.CrossRefPubMedGoogle Scholar
  7. 7.
    Wade, R.H., Chrétien, D., and Job, D. (1990) Characterization of microtubule protofilament numbers. How does the surface lattice accommodate? J. Mol. Biol. 212, 775–786.CrossRefPubMedGoogle Scholar
  8. 8.
    Wade, R.H. and Chrétien, D. (1993) Cryoelectron microscopy of microtubules. J. Struct. Biol. 110, 1–27.CrossRefPubMedGoogle Scholar
  9. 9.
    Mandelkow, E.M., Schultheiss, R., Rapp, R., Muller, M., and Mandelkow, E. (1986) On the surface lattice of microtubules: helix starts, protofilament number, seam, and handedness. J. Cell Biol. 102, 1067–1073.CrossRefPubMedGoogle Scholar
  10. 10.
    Kikkawa, M., Ishikawa, T., Nakata, T., Wakabayashi, T., and Hirokawa, N. (1994) Direct visualization of the microtubule lattice seam both in-vitro and in-vivo. J. Cell Biol. 127, 1965–1971.CrossRefPubMedGoogle Scholar
  11. 11.
    Sosa, H. and Milligan, R.A. (1996) Three-dimensional structure of ncd-decorated microtubules obtained by a back-projection method. J. Mol. Biol. 260, 743–755.CrossRefPubMedGoogle Scholar
  12. 12.
    Kikkawa, M., Okada, Y., and Hirokawa, N. (2000) 15 Å resolution model of the monomeric kinesin motor, KIF1A. Cell 100, 241–252.CrossRefPubMedGoogle Scholar
  13. 13.
    Hirose, K., Akimaru, E., Akiba, T., Endow, A.S., and Amos, L.A. (2006) Large conformational changes in a kinesin motor catalyzed by interaction with microtubules. Mol. Cell 23, 913–923.CrossRefPubMedGoogle Scholar
  14. 14.
    Mitchison, T. and Kirschner, M. (1984) Microtubule assembly nucleated by isolated centrosomes. Nature 312, 232–237.CrossRefPubMedGoogle Scholar
  15. 15.
    Murphy, D.B. and Borisy, G.G. (1975) Association of high-molecular-weight proteins with microtubules and their role in microtubule assembly in vitro. Proc. Natl. Acad. Sci. USA 72, 2696–2700.CrossRefPubMedGoogle Scholar
  16. 16.
    Castoldi, M. and Popov, A.V. (2003) Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr. Purif. 32, 83–88.CrossRefPubMedGoogle Scholar
  17. 17.
    Chandra, R. and Endow, S.A. (1993) Expression of microtubule motor proteins in bacteria for characterization in in vitro motility assays. Methods Cell Biol. 39, 115–127.CrossRefPubMedGoogle Scholar
  18. 18.
    Lockhart, A., Crevel, I.M.-T.C., and Cross, R.A. (1995) Kinesin and ncd bind through a single head to microtubules and compete for a shared MT binding site. J. Mol. Biol. 249, 763–771.CrossRefPubMedGoogle Scholar
  19. 19.
    Fukami, A. and Adachi, K. (1965) A new method of preparation of a self-perforated micro plastic grid and its application. J. Electron Microsc. (Tokyo) 14, 112–118.Google Scholar
  20. 20.
    DeRosier, D.J. and Moore, P.B. (1970) Reconstruction of three-dimensional images from electron micrographs of structures with helical symmetry. J. Mol. Biol. 52, 355–369.CrossRefPubMedGoogle Scholar
  21. 21.
    Crowther, R.A., Henderson, R., and Smith, J.M. (1996) MRC image processing programs. J. Struct. Biol. 116, 9–16.CrossRefPubMedGoogle Scholar
  22. 22.
    Whittaker, M., Carragher, B.O., and Milligan, R.A. (1995) PHOELIX: a package for semi-automated helical reconstruction. Ultramicroscopy 58, 245–259.CrossRefPubMedGoogle Scholar
  23. 23.
    Toyoshima, C. (2000) Structure determination of tubular crystals of membrane proteins. I. Indexing of diffraction patterns. Ultramicroscopy 84, 1–14.CrossRefPubMedGoogle Scholar
  24. 24.
    Yonekura, K., Toyoshima, C., Maki-Yonekura, S., and Namba, K. (2003) GUI programs for processing individual images in early stages of helical image recon-struction—for high-resolution structure analysis. J. Struct. Biol. 144, 184–194.CrossRefPubMedGoogle Scholar
  25. 25.
    Sosa, H., and Chrétien, D. (1998) Relationship between moiré patterns, tubulin shape, and microtubule polarity. Cell Motil. Cytoskelet. 40, 38–43.CrossRefGoogle Scholar
  26. 26.
    Chrétien, D. and Fuller, S.D. (2000) Microtubules switch occasionally into unfavorable configurations during elongation. J. Mol. Biol. 298, 663–676.CrossRefPubMedGoogle Scholar
  27. 27.
    Tani, K., Sasabe, H., and Toyoshima, C. (1996) A set of computer programs for determining defocus and astigmatism in electron images. Ultramicroscopy 65, 31–44.CrossRefGoogle Scholar
  28. 28.
    Jones, T.A., Zou, J.Y., Cowan, S.W., and Kjeldgaard, M. (1991) Improved methods for building protein models in electron density maps and the location of errors in these models. Acta Crystallogr. A 47(Pt. 2), 110–119.CrossRefPubMedGoogle Scholar
  29. 29.
    Frank, J., Radermacher, M., Penczek, P., Zhu, J., Li, Y., Ladjadj, M., and Leith, A. (1996) SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J. Struct. Biol. 116, 190–199.CrossRefPubMedGoogle Scholar
  30. 30.
    van Heel, M., Harauz, G., Orlova, E.V., Schmidt, R., and Schatz, M. (1996) A new generation of the IMAGIC image processing system. J. Struct. Biol. 116, 17–24.CrossRefPubMedGoogle Scholar
  31. 31.
    Trachtenberg, S. and DeRosier, D.J. (1987) Three-dimensional structure of the frozen-hydrated flagellar filament. The left-handed filament of Salmonella typhimurium. J. Mol. Biol. 195, 581–601.CrossRefPubMedGoogle Scholar
  32. 32.
    Wriggers, W. and Birmanns, S. (2001) Using Situs for flexible and rigid-body fitting of multiresolution single-molecule data. J. Struct. Biol. 133, 193–202.CrossRefPubMedGoogle Scholar
  33. 33.
    Roseman, A.M. (2000) Docking structures of domains into maps from cryoelectron microscopy using local correlation. Acta Crystallogr. D Biol. Crystallogr. 56, 1332–1340.CrossRefPubMedGoogle Scholar
  34. 34.
    Volkmann, N. and Hanein, D. (2003) Docking of atomic models into reconstructions from electron microscopy. Methods Enzymol. 374, 204–225.CrossRefPubMedGoogle Scholar

Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Keiko Hirose
    • 1
  • Linda A. Amos
    • 2
  1. 1.Gene Function Research CenterNational Institute of Advanced Industrial Science and Technology (AIST)TsukubaJapan
  2. 2.MRC Laboratory of Molecular BiologyCambridgeUK

Personalised recommendations