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Negative staining and image classification — powerful tools in modern electron microscopy

Abstract

Vitrification is the state-of-the-art specimen preparation technique for molecular electron microscopy (EM) and therefore negative staining may appear to be an outdated approach. In this paper we illustrate the specific advantages of negative staining, ensuring that this technique will remain an important tool for the study of biological macromolecules. Due to the higher image contrast, much smaller molecules can be visualized by negative staining. Also, while molecules prepared by vitrification usually adopt random orientations in the amorphous ice layer, negative staining tends to induce preferred orientations of the molecules on the carbon support film. Combining negative staining with image classification techniques makes it possible to work with very heterogeneous molecule populations, which are difficult or even impossible to analyze using vitrified specimens.

References

  1. Radermacher M, Wagenknecht T, Verschoor A, Frank J. Three-dimensional reconstruction from a single-exposure, random conical tilt series applied to the 50S ribosomal subunit of Escherichia coli. J Microsc 1987; 146:113–136.

    PubMed  CAS  Google Scholar 

  2. Adrian M, Dubochet J, Lepault J, McDowall AW. Cryoelectron microscopy of viruses. Nature 1984; 308:32–36.

    Article  PubMed  CAS  Google Scholar 

  3. Orlova EV, Rahman MA, Gowen B, Volynski KE, Ashton AC, Manser C, van Heel M, Ushkaryov YA. Structure of alpha-latrotoxin oligomers reveals that divalent cation-dependent tetramers form membrane pores. Nat Struct Biol 2000; 7:48–53.

    Article  PubMed  CAS  Google Scholar 

  4. Stark H, Dube P, Luhrmann R, Kastner B. Arrangement of RNA and proteins in the spliceosomal U1 small nuclear ribonucleoprotein particle. Nature 2001; 409:539–542.

    Article  PubMed  CAS  Google Scholar 

  5. Cheng Y, Zak O, Aisen P, Harrison SC, Walz T. Structure of the human transferrin receptor-transferrin complex. Cell 2004; 116:565–576.

    Article  PubMed  CAS  Google Scholar 

  6. Van Heel M. Angular reconstitution: a posteriori assignment of projection directions for 3D reconstruction. Ultramicroscopy 1987; 21:111–123.

    Article  PubMed  Google Scholar 

  7. Lederkremer GZ, Cheng Y, Petre BM, Vogan E, Springer S, Schekman R, Walz T, Kirchhausen T. Structure of the Sec23p/24p and Sec13p/31p complexes of COPII. Proc Natl Acad Sci USA 2001; 98:10704–10709.

    Article  Google Scholar 

  8. Toth EA, Li Y, Sawaya MR, Cheng Y, Ellenberger T. The crystal structure of the bifunctional primase-helicase of bacteriophage T7. Mol Cell 2003; 12:1113–1123.

    Article  PubMed  CAS  Google Scholar 

  9. Takagi J, Petre BM, Walz T, Springer TA. Global conformational rearrangements in integrin extracellular domains in outside-in and inside-out signaling. Cell 2002; 110:599–611.

    Article  PubMed  CAS  Google Scholar 

  10. Takagi J, Strokovich K, Springer TA, Walz T. Structure of integrin α5β1 in complex with fibronectin. EMBO J 2003; 22:4607–4615.

    Article  PubMed  CAS  Google Scholar 

  11. Leggett DS, Hanna J, Borodovsky A, Crosas B, Schmidt M, Baker RT, Walz T, Ploegh H, Finley D. Multiple associated proteins regulate proteasome structure and function. Mol Cell 2002; 10:495–507.

    Article  PubMed  CAS  Google Scholar 

  12. Cascio P, Call M, Petre BM, Walz T, Goldberg AL. Properties of the hybrid form of the 26S proteasome containing both 19S and PA28 complexes. EMBO J 2002; 21:2636–2645.

    Article  PubMed  CAS  Google Scholar 

  13. Walz T, Jamieson SJ, Bowers CM, Bullough PA, Hunter CN. Projection structures of three photosynthetic complexes from Rhodobacter sphaeroides: LH2 at 6 Å, LH1 and RC-LH1 at 25 Å. J Mol Biol 1998; 282:833–845.

    Article  PubMed  CAS  Google Scholar 

  14. Frank J, Radermacher M, Penczek P, Zhu J, Li Y, Ladjadj M, Leith A. SPIDER and WEB: processing and visualization of images in 3D electron microscopy and related fields. J Struct Biol 1996; 116:190–199.

    Article  PubMed  CAS  Google Scholar 

  15. Adrian M, Dubochet J, Fuller SD, Harris JR. Cryo-negative staining. Micron 1998; 29:145–160.

    Article  PubMed  CAS  Google Scholar 

  16. Golas MM, Sander B, Will CL, Lurhmann R, Stark H. Molecular architecture of the multiprotein splicing factor SF3b. Science 2003; 300:980–984.

    Article  PubMed  CAS  Google Scholar 

  17. Bremer A, Henn C, Engel A, Baumeister W, Aebi U. Has negative staining still a place in biomacromolecular electron microscopy? Ultramicroscopy 1992; 46:85–111.

    Article  PubMed  CAS  Google Scholar 

  18. Baumeister W, Dahlmann B, Hegerl R, Kopp F, Kuehn L, Pfeifer G. Electron microscopy and image analysis of the multicatalytic proteinase. FEBS Lett 1988; 241:239–245.

    Article  PubMed  CAS  Google Scholar 

  19. Brink J, Van Breemen JF, Keegstra W, Van Bruggen EF. Computer image analysis of two-dimensional crystals of beef heart NADH: ubiquinone oxidoreductase fragments. I. Comparison of crystal structures in various negative stains. Ultramicroscopy 1989; 27:79–90.

    Article  PubMed  CAS  Google Scholar 

  20. Walz T, Haner M, Wu XR, Henn C, Engel A, Sun TT, Aebi U. Towards the molecular architecture of the asymmetric unit membrane of the mammalian urinary bladder epithelium: a closed “twisted ribbon” structure. J Mol Biol 1995; 248:887–900.

    Article  PubMed  CAS  Google Scholar 

  21. Zhao FQ, Craig R. Capturing time-resolved changes in molecular structure by negative staining. J Struct Biol 2003; 141:43–52.

    Article  PubMed  CAS  Google Scholar 

  22. Frank J. Three-Dimensional Electron Microscopy of Macromolecular Assemblies. 1st ed. San Diego: Academic Press, Inc.; 1996.

    Google Scholar 

  23. Samso M, Palumbo MJ, Radermacher M, Liu JS, Lawrence CE. A Bayesian method for classification of images from electron micrographs. J Struct Biol 2002; 138:157–170.

    Article  PubMed  Google Scholar 

  24. van Heel M, Harauz G, Orlova EV, Schmidt R, Schatz M. A new generation of the IMAGIC image processing system. J Struct Biol 1996; 116:17–24.

    Article  PubMed  Google Scholar 

  25. Ludtke SJ, Baldwin PR, Chiu W. EMAN: semiautomated software for high-resolution single-particle reconstructions. J Struct Biol 1999; 128:82–97.

    Article  PubMed  CAS  Google Scholar 

  26. Groll M, Ditzel L, Lowe J, Stock D, Bochtler M, Bartunik HD, Huber R. Structure of 20S proteasome from yeast at 2.4 Å resolution. Nature 1997; 386:463–471.

    Article  PubMed  CAS  Google Scholar 

  27. Bi X, Corpina RA, Goldberg J. Structure of the Sec23/24-Sar1 pre-budding complex of the COPII vesicle coat. Nature 2002; 419:271–277.

    Article  PubMed  CAS  Google Scholar 

  28. Adams GM, Crotchett B, Slaughter CA, DeMartino GN, Gogol EP. Formation of proteasome-PA700 complexes directly correlates with activation of peptidase activity. Biochemistry 1998; 37:12927–12932.

    Article  PubMed  CAS  Google Scholar 

  29. Stauffer KA, Hoenger A, Engel A. Two-dimensional crystals of Escherichia coli maltoporin and their interaction with the maltose-binding protein. J Mol Biol 1992; 223:1155–1165.

    Article  PubMed  CAS  Google Scholar 

  30. Stahlberg H, Dubochet J, Vogel H, Ghosh R. Are the light-harvesting I complexes from Rhodospirillum rubrum arranged around the reaction centre in a square geometry? J Mol Biol 1998; 282:819–831.

    Article  PubMed  CAS  Google Scholar 

  31. Tahara Y, Ohnishi S, Fujiyoshi Y, Kimura Y, Hayashi Y. A pH induced two-dimensional crystal of membrane-bound Na+,K+-ATPase of dog kidney. FEBS Lett 1993; 320:17–22.

    Article  PubMed  CAS  Google Scholar 

  32. Tahara Y, Oshima A, Hirai T, Mitsuoka K, Fujiyoshi Y, Hayashi Y. The 11 Å resolution projection map of Na+/K+-ATPase calculated by application of single particle analysis to two-dimensional crystal images. J Electron Microsc 2000; 49:583–587.

    CAS  Google Scholar 

  33. Xiong JP, Stehle T, Diefenbach B, Zhang R, Dunker R, Scott DL, Joachimiak A, Goodman SL, Arnaout MA. Crystal structure of the extracellular segment of integrin αvβ3. Science 2001; 294:339–345.

    Article  PubMed  CAS  Google Scholar 

  34. Leahy DJ, Aukhil I, Erickson HP. 2.0 Å crystal structure of a four-domain segment of human fibronectin encompassing the RGD loop and synergy region. Cell 1996; 84:155–164.

    Article  PubMed  CAS  Google Scholar 

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Correspondence to Thomas Walz.

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Published: March 19, 2004.

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Ohi, M., Li, Y., Cheng, Y. et al. Negative staining and image classification — powerful tools in modern electron microscopy. Biol. Proced. Online 6, 23–34 (2004). https://doi.org/10.1251/bpo70

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  • DOI: https://doi.org/10.1251/bpo70

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