Advertisement

Low Dose Techniques and Cryo-Electron Microscopy

Protocol
First Online:
Part of the Methods in Molecular Biology book series (MIMB, volume 955)

Abstract

Our understanding of subcellular structures has been greatly increased owing to electron microscopy, even though radiation damage of biological samples by the electron beam demanded staining techniques. Technological and instrumental advances of electron microscopy have, however, established various highly sophisticated techniques to study biological systems in their native states without staining and thus facilitated comprehension of rather intact structures of biological components. Among these techniques, electron crystallography is a well-established one to analyze membrane protein structures within lipid bilayers, without staining at close-to-physiological conditions. Structures of membrane proteins could be analyzed at resolutions better than 3Å by electron crystallography due to techniques of low dose and cryo-electron microscopy (cryo-EM). Here, recent cryo-EM technological and instrumental advances crucial to optimal data collection in electron crystallography are summarized as well as examples of structures of membrane proteins analyzed with the help of this method.

Key words

Cryo-EM Low-dose Multiple dose system Electron crystallography Beam damage 
This is a preview of subscription content, log in to check access.

Notes

Acknowledgments

These studies were performed in wonderful collaborations with many researchers whose names were recorded as authors in each referenced paper. This work was supported by Grants-in-Aid for Scientific Research (S) and the Japan New Energy and Industrial Technology Development Organization (NEDO).

References

  1. 1.
    Henderson R, Unwin PNT (1975) Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257:28–32PubMedCrossRefGoogle Scholar
  2. 2.
    Unwin PNT, Henderson R (1975) Molecular structure determination by electron microscopy of unstained crystalline specimens. J Mol Biol 94:425–440PubMedCrossRefGoogle Scholar
  3. 3.
    Kuo IAM, Glaeser RM (1975) Development of methodology for low-dose exposure, high-resolution electron microscopy of biological specimens. Ultramicroscopy 1:53–66PubMedCrossRefGoogle Scholar
  4. 4.
    Henderson R, Baldwin JM, Ceska TA, Zemlin F, Beckmann E, Downing KH (1990) Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J Mol Biol 213:899–929PubMedCrossRefGoogle Scholar
  5. 5.
    Uyeda N, Kobayashi T, Ishizuka K, Fujiyoshi Y (1999) High Voltage Electron Microscopy for Image Discrimination of Constituent Atoms in Crystals and Molecules. Chemia Scripta 14:47–61Google Scholar
  6. 6.
    Fujiyoshi Y, Kobayashi T, Ishizuka K, Uyeda N, Ishida Y, Hrada Y (1980) Digital reconstruction of bright field phase contrast images from high resolution electron micrographs. Ultramicroscopy 5:459–468Google Scholar
  7. 7.
    Uyeda N, Kobayashi T, Ishizuka K, Fujiyoshi Y (1980) Crystal structure of Ag TCNQ. Nature 285:95–97CrossRefGoogle Scholar
  8. 8.
    Adrian M, Dubochet J, Lepault J, McDowall AW (1984) Cryo-electron microscopy of viruses. Nature 308:32–37PubMedCrossRefGoogle Scholar
  9. 9.
    Tani K, Mitsuma T, Hiroaki Y, Kamegawa A, Nishikawa K, Tanimura Y, Fujiyoshi Y (2009) Mechanism for aquaporin’s fast and selective water conduction and proton exclusion. J Mol Biol 389:694–706PubMedCrossRefGoogle Scholar
  10. 10.
    Fujiyoshi Y (1998) The structural study of membrane proteins by electron crystallography. Adv Biophys 35:25–80PubMedCrossRefGoogle Scholar
  11. 11.
    Hirai T, Murata K, Mitsuoka K, Kimura Y, Fujiyoshi Y (1999) Trehalose embedding technique for high resolution electron crystallography: application to structural study on bacteriorhodopsin. J Electron Microsc 48:653–658CrossRefGoogle Scholar
  12. 12.
    Fujiyoshi Y, Mizusaki T, Morikawa K, Yamagishi H, Aoki Y, Kihara H, Harada Y (1991) Development of a superfluid helium stage for high-resolution cryo ­electron microscopy. Ultramicroscopy 38:241–251CrossRefGoogle Scholar
  13. 13.
    Kimura Y, Vassylyev DG, Miyazawa A, Kidera A, Matsushima M, Mitsuoka K, Murata K, Hirai T, Fujiyoshi Y (1997) Surface of ­bacteriorhodopsin revealed by high-resolution electron crystallography. Nature 389:206–211PubMedCrossRefGoogle Scholar
  14. 14.
    Gyobu N, Tani K, Hiroaki Y, Kmegawa A, Mitsuoka K, Fujiyoshi Y (2004) Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique. J Struct Biol 146:325–333PubMedCrossRefGoogle Scholar
  15. 15.
    Gonen T, Cheng Y, Sliz P, Hiroaki Y, Fujiyoshi Y, Harrison SC, Walz T (2005) Aquaporin-0 membrane junctions reveal the structure of a closed water pore. Nature 438:633–638PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  1. 1.Department of Basic Biology, Cellular and Structural Physiology Institute (CeSPI)Nagoya UniversityNagoyaJapan

Personalised recommendations

Cite protocol

Buy options