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.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsReferences
Henderson R, Unwin PNT (1975) Three-dimensional model of purple membrane obtained by electron microscopy. Nature 257:28–32
Unwin PNT, Henderson R (1975) Molecular structure determination by electron microscopy of unstained crystalline specimens. J Mol Biol 94:425–440
Kuo IAM, Glaeser RM (1975) Development of methodology for low-dose exposure, high-resolution electron microscopy of biological specimens. Ultramicroscopy 1:53–66
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 213:899–929
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 14:47–61
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 5:459–468
Uyeda N, Kobayashi T, Ishizuka K, Fujiyoshi Y (1980) Crystal structure of Ag TCNQ. Nature 285:95–97
Adrian M, Dubochet J, Lepault J, McDowall AW (1984) Cryo-electron microscopy of viruses. Nature 308:32–37
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 389:694–706
Fujiyoshi Y (1998) The structural study of membrane proteins by electron crystallography. Adv Biophys 35:25–80
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 48:653–658
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 38:241–251
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 389:206–211
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 146:325–333
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 438:633–638
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).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media New York
About this protocol
Cite this protocol
Fujiyoshi, Y. (2013). Low Dose Techniques and Cryo-Electron Microscopy. In: Schmidt-Krey, I., Cheng, Y. (eds) Electron Crystallography of Soluble and Membrane Proteins. Methods in Molecular Biology, vol 955. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-176-9_6
Download citation
DOI: https://doi.org/10.1007/978-1-62703-176-9_6
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-175-2
Online ISBN: 978-1-62703-176-9
eBook Packages: Springer Protocols