Abstract
Single particle analysis in electron microscopy allows direct observation of the reconstructed three-dimensional structures of protein molecules. This method enables a more comprehensive study of membrane proteins which have been problematic in structural studies using X-ray crystallography. These membrane proteins include the voltage-sensitive ion channel proteins, which play an important rule in neural activities, and have great medical significance. The method described is supported by the development of cryo-electron microscopy and the angular reconstitution method. This review summarizes certain principles governing single particle analysis employing angular reconstitution. This method was applied to our study of the voltage-sensitive sodium channel, and the results are discussed. With improvements in resolutions and statistical analyses, the single particle technique is considered to be advantageous in studies of the structural changes and molecular interactions of protein molecules.
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References
- 1.DeRosier D.J., & Klug A. (1968) Reconstruction of three-demensional structures from electron micrographs. Nature, 217, 130–134. [DOI] [PubMed] [Google Scholar]
- 2.Unwin P.N.T., & Henderson R. (1975) Molecular structure determination by electron microscopy of unstained crystalline specimens. J. Mol. Biol., 94, 425–440. [DOI] [PubMed] [Google Scholar]
- 3.Bottcher B., Wynne S.A., & Crowther R.A. (1997) Determination of the fold of the core protein of hepatitis B virus by electron cryomicroscopy. Nature, 386, 88–91. [DOI] [PubMed] [Google Scholar]
- 4.Frank J., & Agrawal R.K. (2000) A ratchet-like inter-subunit reorganization of the ribosome during translocation. Nature, 406, 318–322. [DOI] [PubMed] [Google Scholar]
- 5.van Heel M., Gowen B., Matadeen R., Orlova E.V., Finn R., Pape T., Cohen D., Stark H., Schmiidt R., Schats M., & Patwardhan A. (2000) Single-particle electron cryomicroscopy: towards atomic resolution. Qual Rev Biophys, 33, 307–369. [DOI] [PubMed] [Google Scholar]
- 6.Fujiyoshi Y. (1998) The structural study of membrane proteins by electron crystallography. Adv. Biophys., 35, 25–80. [DOI] [PubMed] [Google Scholar]
- 7.Serysheva II, Orlova E.V., Chiu W., Sherman M.B., Hamilton S.L., & van Heel M. (1995) Electron cryomicroscopy and angular reconstitution used to visualize the skeletal muscle calcium release channel. Nat. Struct. Biol., 2, 18–24. [DOI] [PubMed] [Google Scholar]
- 8.Sato C., Ueno Y., Asai K., Takahashi K., Sato M., Engel A., & Fujiyoshi Y. (2001) The voltage-sensitive sodium channel is a bell-shaped molecule with several cavities. Nature, 409, 1047–1051. [DOI] [PubMed] [Google Scholar]
- 9.Murata K., Odahara N., Kuniyasu A., Sato Y., Nakayama H., & Nagayama K. (2001) Asymmetric arrangement of auxiliary subunits of skeletal muscle voltage-gated l-type Ca(2+) channel. Biochem. Biophys. Res. Commun., 282, 284–291. [DOI] [PubMed] [Google Scholar]
- 10.Sokolova O., Kolmakova-Partensky L., & Grigorieff N. (2001) Three-Dimensional Structure of a Voltage-Gated Potassium Channel at 2.5 nm Resolution. Structure, 9, 215–220. [DOI] [PubMed] [Google Scholar]
- 11.Doyle D.A., Morais Cabral J., Pfuetzner R.A., Kuo A., Gulbis J.M., Cohen S.L., Chait B.T., & MacKinnon R. (1998) The structure of the potassium channel: molecular basis of K+ conduction and selectivity. Science, 280, 69–77. [DOI] [PubMed] [Google Scholar]
- 12.Frank J. (1992) Electron tomography: three-dimensional imaging with the transmission electron microscope. Plenum Press, NewYork. [Google Scholar]
- 13.Frank J. (1996) Three-dimentional electron microscopy of macromolecular assemblies. Academic Press, Inc., California. [Google Scholar]
- 14.Adrian M., Dubochet J., Lepault J., & McDowall A.W. (1984) Cryo-electron microscopy of viruses. Nature, 308, 32–36. [DOI] [PubMed] [Google Scholar]
- 15.Fujiyoshi Y., Morikawa K., Uyeda N., Ozeki H., & Yamagishi H. (1983) Electron microscopy of tRNA crystals. I. Thin crystals negatively stained with uranyl acetate. Ultramicroscopy, 12, 210–212. [PubMed] [Google Scholar]
- 16.Katayama E., Funahashi H., Michikawa T., Shiraishi T., Ikemoto T., Iino M., & Mikoshiba K. (1996) Native structure and arrangement of inositol-1,4,5-trisphosphate receptor molecules in bovine cerebellar Purkinje cells as studied by quick-freeze deep-etch electron microscopy. EMBO J., 15, 4844–4851. [PMC free article] [PubMed] [Google Scholar]
- 17.Penczek P., Radermacher M., & Frank J. (1992) Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy, 40, 33–53. [PubMed] [Google Scholar]
- 18.van Heel M. (1989) Classification of very large electron microscopical image data sets. Optik, 82, 114–126. [Google Scholar]
- 19.Pascual A., Barcena M., Merelo J.J., & Carazo J.M. (2000) Mapping and fuzzy classification of macromolecular images using self-organizing neural networks. Ultramicroscopy, 84, 85–99. [DOI] [PubMed] [Google Scholar]
- 20.Penczek P.A., Zhu J., & Frank J. (1996) A common-lines based method for determining orientations for N > 3 particle projections simultaneously. Ultramicroscopy, 63, 205–218. [DOI] [PubMed] [Google Scholar]
- 21.Grigorieff N. (1998) Three-dimensional structure of bovine NADH:ubiquinone oxidoreductase (complex I) at 22 Å in ice. J. Mol. Biol., 277, 1033–1046. [DOI] [PubMed] [Google Scholar]
- 22.Catterall W.A. (2000) From ionic currents to molecular mechanisms: the structure and function of voltage-gated sodium channels. Neuron, 26, 13–25. [DOI] [PubMed] [Google Scholar]
- 23.Deschenes I., Baroudi G., Berthet M., Barde I., Chalvidan T., Denjoy I., Guicheney P., & Chahine M. (2000) Electrophysiological characterization of SCN5A mutations causing long QT (E1784K) and Brugada (R1512W and R1432G) syndromes. Cardiovasc. Res., 46, 55–65. [DOI] [PubMed] [Google Scholar]
- 24.Miller J.A., Agnew W.S., & Levinson S.R. (1983) Principal glycopeptide of the tetrodotoxin/saxitoxin binding protein from Electrophorus electricus: isolation and partial chemical and physical characterization. Biochemistry, 22, 462–470. [DOI] [PubMed] [Google Scholar]
- 25.Sato C., Sato M., Iwasaki A., Doi T., & Engel A. (1998) The sodium channel has four domains surrounding a central pore. J Struct Biol, 121, 314–325. [DOI] [PubMed] [Google Scholar]
- 26.Ellisman M.H., Miller J.A., & Agnew W.S. (1983) Molecular morphology of the tetrodotoxin-binding sodium channel protein from Electrophorus electricus in solubilized and reconstituted preparations. J. Cell. Biol., 97, 1834–1840. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 27.Heinemann S.H., Terlau H., Stuhmer W., Imoto K., & Numa S. (1992) Calcium channel characteristics conferred on the sodium channel by single mutations. Nature, 356, 441–443. [DOI] [PubMed] [Google Scholar]
- 28.Stuhmer W., Conti F., Suzuki H., Wang X.D., Noda M., Yahagi N., Kubo H., & Numa S. (1989) Structural parts involved in activation and inactivation of the sodium channel. Nature, 339, 597–603. [DOI] [PubMed] [Google Scholar]
- 29.Orlova E.V., Serysheva II, van Heel M., Hamilton S.L., & Chiu W. (1996) Two structural configurations of the skeletal muscle calcium release channel. Nat. Struct. Biol., 3, 547–552. [DOI] [PubMed] [Google Scholar]
- 30.Remigy H.W., Stahlberg H., Fotiadis D., Muller S.A., Wolpensinger B., Engel A., Hauska G., & Tsiotis G. (1999) The reaction center complex from the green sulfur bacterium Chlorobium tepidum: a structural analysis by scanning transmission electron microscopy. J. Mol. Biol., 290, 851–858. [DOI] [PubMed] [Google Scholar]
