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Applications of Electron Cryo-Microscopy to Cardiovascular Research

  • Ashraf Kitmitto
Part of the Methods in Molecular Medicine book series (MIMM, volume 129)

Abstract

Electron cryo-microscopy (cryo-EM) techniques have wide applications for the study of biological structures. The focus of this chapter is the use of cryo-EM and associated methods for the analysis of the three-dimensional (3D) structure of proteins and multicomponent macromolecular assemblies. Data evolving from these methods pertaining to the quaternary organization of proteins and protein-protein interactions, bridges an important gap between linear genomic information and understanding physiological function. This chapter provides general methods for examining two-dimensional crystalline arrays of proteins as well as single, randomly orientated protein complexes. It is significant that single-particle analysis of electron microscopy images has provided the only 3D data to-date for the two principal components of muscle excitation contraction coupling in the heart, namely the L-type voltage-gated calcium channel and the ryanodine receptor. This chapter describes approaches for identifying the extracellular and intracellular domains of the 3D structure of the L-type voltage-gated calcium channel and also incorporates general details for labeling and visualizing His-tagged proteins.

Key Words

Cryo-electron microscopy electron crystallography single particle analysis L-type voltage-gated calcium channels cardiac calcium cycling 

References

  1. 1.
    Sharma, M. R., Jeyakumar, L. H., Fleischer, S., and Wagenknecht, T. (2000) Three-dimensional structure of ryanodine receptor isoform three in two conformational states as visualized by cryo-electron microscopy. J. Biol. Chem. 275, 9485–9491.CrossRefPubMedGoogle Scholar
  2. 2.
    Samso, M. and Wagenknecht, T. (2002) Apocalmodulin and Ca2+-calmodulin bind to neighboring locations on the ryanodine receptor. J. Biol. Chem. 277, 1349–1353.CrossRefPubMedGoogle Scholar
  3. 3.
    Unger, V. M., Kumar, N. M., Gilula, N. B., and Yeager, M. (1999) Electron cryocrystallography of a recombinant cardiac gap junction channel, in Gap Junction-Mediated Intercellular Signalling in Health and Disease. pp. 22–37.Google Scholar
  4. 4.
    Rubinstein, J. L., Walker, J. E., and Henderson, R. (2003) Structure of the mitochondrial ATP synthase by electron cryomicroscopy. EMBO 22, 6182–6192.CrossRefGoogle Scholar
  5. 5.
    Grigorieff, N. (1998) Three-dimensional structure of bovine NADH: ubiquinone oxidoreductase (Complex I) at 22 angstrom in ice. J. Mol. Biol. 277, 1033–1046.CrossRefPubMedGoogle Scholar
  6. 6.
    MacLennan, D. H., Abu-Abed, M., and Kang, C. (2002) Structure-function relationships in Ca2+ cycling proteins. J. Mol.Cell. Cardiol. 34, 897–918.CrossRefPubMedGoogle Scholar
  7. 7.
    Toyoshima, C., Nakasako, M., Nomura, H., and Ogawa, H. (2000) Crystal structure of the calcium pump of sarcoplasmic reticulum at 2.6 angstrom resolution. Nature 405, 647–655.CrossRefPubMedGoogle Scholar
  8. 8.
    Sharma, M. R., Penczek, P., Grassucci, R., Xin, H. B., Fleischer, S., and Wagenknecht, T. (1998) Cryoelectron microscopy and image analysis of the cardiac ryanodine receptor. J. Biol. Chem. 273, 18,429–18,434.CrossRefPubMedGoogle Scholar
  9. 9.
    Wang, M. C., Collins, R. F., Ford, R. C., Berrow, N. S., Dolphin, A. C., and Kitmitto, A. (2004) The three-dimensional structure of the cardiac L-type voltage-gated calcium channel—comparison with the skeletal muscle form reveals a common architectural motif. J. Biol. Chem. 279, 7159–7168.CrossRefPubMedGoogle Scholar
  10. 10.
    Sharma, M. R., Jeyakumar, L. H., Fleischer, S., and Wagenknecht, T. (2002) Three-dimensional visualization of FKBP12.6 binding to cardiac ryanodine receptor (RyR2) in open buffer condition. Biophys. J. 82, 3145.Google Scholar
  11. 11.
    Berriman, J. and Unwin, N. (1994) Analysis of transient structures by cryomicroscopy combined with rapid mixing of spray droplets. Ultramicroscopy 56, 241–252.CrossRefPubMedGoogle Scholar
  12. 12.
    Brickley, K., Campbell, V., Berrow, N., et al. (1995) Use of site-directed antibodies to probe the topography of the alpha(2) subunit of voltage-gated Ca2+ channels. FEBS Lett. 364, 129–133.CrossRefPubMedGoogle Scholar
  13. 13.
    Adrian, M., Dubochet, J., Fuller, S. D., and Harris, J. R. (1998) Cryo-negative staining. Micron. 29, 145–160.CrossRefPubMedGoogle Scholar
  14. 14.
    Collins, R. F., Frye, S. A., Kitmitto, A., Ford, R. C., Tonjum, T., and Derrick, J. P. (2004) Structure of the Neisseria meningitidis outer membrane PilQ secretin complex at 12 angstrom resolution. J. Biol. Chem. 279, 39,750–39,756.CrossRefPubMedGoogle Scholar
  15. 15.
    Florio, V., Striessnig, J., and Catterall, W. A. (1992) Purification and reconstitution of skeletal-muscle calcium Channels. Methods Enzymol. 207, 529–546.CrossRefPubMedGoogle Scholar
  16. 16.
    Wang, M. C., Dolphin, A., and Kitmitto, A. (2004) L-type voltage-gated calcium channels: understanding function through structure. FEBS Lett. 564, 245–250.CrossRefPubMedGoogle Scholar
  17. 17.
    Buchel, C., Morris, E., Orlova, E., and Barber, J. (2001) Localisation of the PsbH subunit in photosystem II: a new approach using labeling of His-tags with a Ni2+-NTA gold cluster and single particle analysis. J. Molec. Biol. 312, 371–379.CrossRefPubMedGoogle Scholar
  18. 18.
    Hainfeld, J. F., Liu, W. Q., Halsey, C. M. R., Freimuth, P., and Powell, R. D. (1999) Ni-NTA-gold clusters target his-tagged proteins. J. Struct. Biol. 127, 185–198.CrossRefPubMedGoogle Scholar
  19. 19.
    Vonck, J. (2000) Parameters affecting specimen flatness of two-dimensional crystals for electron crystallography. Ultramicroscopy 85, 123–129.CrossRefPubMedGoogle Scholar
  20. 20.
    De Carlo, S., Adrian, H., Kallin, P., Mayer, J. M., and Dubochet, J. (1999) Unexpected property of trehalose as observed by cryo-electron microscopy. J. Microscop. Oxford 196, 40–45.CrossRefGoogle Scholar
  21. 21.
    Benacquista, B. L., Sharma, M. R., Samso, M., Zorzato, F., Treves, S., and Wagenknecht, T. (2000) Amino acid residues 4425–4621 localized on the three-dimensional structure of the skeletal muscle ryanodine receptor. Biophy. J. 78, 1349–1358.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Ashraf Kitmitto
    • 1
  1. 1.School of MedicineUniversity of ManchesterUK

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