Hostname: page-component-586b7cd67f-rcrh6 Total loading time: 0 Render date: 2024-11-27T14:42:12.297Z Has data issue: false hasContentIssue false
  • English
  • Français

On the Experimental Use of Light Metal Salts for Negative Staining

Published online by Cambridge University Press:  03 March 2008

William H. Massover
William H. Massover
Affiliation:
Department of Biological Sciences, Rutgers University–Newark, Newark, NJ 07102, USA
Get access

Abstract

All common negative stains are salts of heavy metals. To remedy several technical defects inherent in the use of heavy metal compounds, this study investigates whether salts of the light metals sodium, magnesium, and aluminum can function as negative stains. Screening criteria require aqueous solubility at pH 7.0, formation of a smooth amorphous layer upon drying, and transmission electron microscope imaging of the 87-Å (8.7-nm) lattice periodicity in thin catalase crystals. Six of 23 salts evaluated pass all three screens; detection of the protein shell in ferritin macromolecules indicates that light metal salts also provide negative staining of single particle specimens. Appositional contrast is less than that given by heavy metal negative stains; image density can be raised by increasing electron phase contrast and by selecting salts with phosphate or sulfate anions, thereby adding strong scattering from P or S atoms. Low-dose electron diffraction of catalase crystals negatively stained with 200 mM magnesium sulfate shows Bragg spots extending out to 4.4 Å. Future experimental use of sodium phosphate buffer and magnesium sulfate for negative staining is anticipated, particularly in designing new cocktail (multicomponent) negative stains able to support and protect protein structure to higher resolution levels than are currently achieved.

Keywords

aluminum lactatecatalaseelectron microscopyferritinmagnesium sulfatenegative stainingprotein structuresodium phosphatestructural biology
Type
Research Article
Information
Microscopy and Microanalysis , Volume 14 , Issue 2 , April 2008 , pp. 126 - 137
Copyright
© 2008 Microscopy Society of America

Access options

Get access to the full version of this content by using one of the access options below. (Log in options will check for institutional or personal access. Content may require purchase if you do not have access.)

References

REFERENCES

Adrian, M., Dubochet, J., Fuller, S.D. & Harris, J.R. (1998). Cryo-negative staining. Micron 29, 145–160.Google Scholar
Böttcher, C., Ludwig, K., Herrmann, A., Van Heel, M. & Stark, H. (1999). Structure of influenza haemagglutinin at neutral and at fusogenic pH by electron cryo-microscopy. FEBS Lett 463, 255–259.Google Scholar
Bradley, D.E. (1962). A study of the negative staining process. J Gen Microbiol 29, 503–516.Google Scholar
Bremer, A., Henn, C., Engel, A., Baumeister, W. & Aebi, U. (1992). Has negative staining still a place in macromolecular electron microscopy? Ultramicroscopy 46, 85–111.Google Scholar
Chiu, W., Baker, M.L., Jiang, W., Dougherty, M. & Schmid, M.F. (2005). Electron cryomicroscopy of biological machines at subnanometer resolution. Structure 13, 353–372.Google Scholar
Cohen, H.A., Chiu, W. & Hosoda, J. (1981). Reconstruction of two-dimensional projections of GP 32*1. In Proceedings of the 39th Annual Meeting of the Electron Microscopy Society of America, Bailey, G.W. (Ed.), pp. 38–39. Baton Rouge, LA: Claitor's Publication Division.
De Carlo, S., El-Bez, C., Alvarez-Rúa, C., Borge, J. & Dubochet, J. (2002). Cryo-negative staining reduces electron beam sensitivity of vitrified biological particles. J Struct Biol 138, 216–226.Google Scholar
El-Bez, C., Adrian, M., Dubochet, J. & Cover, T.L. (2005). High resolution structural analysis of Helicobacter pylori VacA toxin oligomers by cryo-negative staining electron microscopy. J Struct Biol 151, 215–228.Google Scholar
Flanagan, J.M., Wall, J.S., Capel, M.S., Schneider, D.K. & Shanklin, J. (1995). Scanning transmission electron microscopy and small-angle scattering provide evidence that native Escherichia coli ClpP is a tetramer with an axial pore. Biochem 34, 10910–10917.Google Scholar
Gerle, C., Tani, K., Yokoyama, K., Tamakoshi, M., Yoshida, M., Fujiyoshi, Y. & Mitsuoka, K. (2006). Two-dimensional crystallization and analysis of projection images of intact Thermus thermophilus V-ATPase. J Struct Biol 153, 200–206.Google Scholar
Glaeser, R.M., Downing, K., Derosier, D., Chiu, W. & Frank, J. (2007). Electron Crystallography of Biological Macromolecules. New York: Oxford University Press.
Gonen, T., Cheng, Y., Sliz, P., Hiroaki, Y., Fujiyoshi, Y., Harrison, S.C. & Walz, T. (2005). Lipid–protein interactions in double-layered two-dimensional AQP0 crystals. Nature 438, 633–638.Google Scholar
Hainfeld, J.F., Safer, D., Wall, J.S., Simon, M., Lin, B. & Powell, R.D. (1994). Methylamine vanadate (NanoVan) negative stain. In Proceedings of the 52nd Annual Meeting of the Electron Microscopy Society of America, Bailey, G.W. & Garratt-Reed, A.J. (Eds.), pp. 133–134. San Francisco: San Francisco Press, Inc.
Harris, J.R. (1997). Negative Staining and Cryoelectron Microscopy. Oxford: BIOS Publishers.
Harris, J.R., Gerber, M., Gebauer, W., Wernicke, W. & Markl, J. (1996). Negative stains containing trehalose. Application to tubular and filamentous structures. J Microsc Soc Am 2, 43–52.Google Scholar
Harris, J.R. & Horne, R.W. (1994). Negative staining: A brief assessment of current technical benefits, limitations and future possibilities. Micron 25, 5–13.Google Scholar
Harris, J.R. & Scheffler, D. (2002). Routine preparation of air-dried negatively stained and unstained specimens on holey carbon support films: A review of applications. Micron 33, 503–516.Google Scholar
Hoenger, A. & Aebi, U. (1996). 3-D reconstructions from ice-embedded and negatively stained biomacromolecular assemblies: A critical comparison. J Struct Biol 117, 99–116.Google Scholar
Horne, R.W. & Wildy, P. (1979). An historical account of the development and applications of the negative staining technique to the electron microscopy of viruses. J Microsc 117, 103–122.Google Scholar
Imai, H., Narita, A., Schroer, T.A. & Maeda, Y. (2006). Two-dimensional averaged images of the dynactin complex revealed by single particle analysis. J Mol Biol 359, 833–839.Google Scholar
Jésior, J.-C. (1982). The grid sectioning technique: A study of catalase platelets. EMBO J 1, 1423–1428.Google Scholar
Massover, W.H. (1993). Ultrastructure of ferritin and apoferritin: A review. Micron 24, 389–437.Google Scholar
Massover, W.H. (1995). New dimensions for negative staining in electron microscopy. Acta Microscopica 4(Suppl. A), xxiii.Google Scholar
Massover, W.H. (2000). Magnesium acetate: A divalent light-atom negative stain. Microsc Microanal 6(Suppl. 2), 478–479.Google Scholar
Massover, W.H. (2004). A low-dose electron diffraction assay for protection of protein structure against damage from drying. Microsc Microanal 10, 261–268.Google Scholar
Massover, W.H. (2005). Magnesium sulfate: A dual scattering unconventional negative stain. Microsc Microanal 11(Suppl. 2), 542CD–543CD.Google Scholar
Massover, W.H. (2006a). Bubbling in light-atom salts: A new method for direct evaluation of electron beam-induced radiation damage. Microsc Microanal 12(Suppl. 2), 390CD–391CD.Google Scholar
Massover, W.H. (2006b). A new method for directly evaluating protective additives acting against electron beam-induced radiation damage. In Proceedings of the 16th International Microscopy Congress, Sapporo, Japan, Vol. I, p. 493. [http://www.imclb.jp/]
Massover, W.H. (2007). Radiation damage to protein specimens from electron beam imaging and diffraction: A mini-review of anti-damage approaches, with special reference to synchrotron X-ray crystallography. J Synchrotron Radiat 14, 116–127.Google Scholar
Massover, W.H., Lai, P.F. & Marsh, P. (2001). Negative staining permits 4.0 Å resolution with low-dose electron diffraction of catalase crystals. Ultramicroscopy 90, 7–12.Google Scholar
Massover, W.H. & Marsh, P. (1997). Unconventional negative stains: Heavy metals are not required for negative staining. Ultramicroscopy 69, 139–150.Google Scholar
Massover, W.H. & Marsh, P. (2000). Light atom derivatives of structure preserving sugars are unconventional negative stains. Ultramicroscopy 85, 107–121.Google Scholar
Meissner, U., Schroder, E., Scheffler, D., Martin, A.G. & Harris, J.R. (2007). Formation, TEM study and 3D reconstitution of the human erythrocyte peroxiredoxin-2 dodecahedral higher-order assembly. Micron 38, 29–39.Google Scholar
Ohi, M., Li, Y., Cheng, Y. & Walz, T. (2004). Negative staining and image classification—Powerful tools in modern electron microscopy. Biol Proc Online 6, 23–34.Google Scholar
Puri, T., Wendler, P., Sigala, B., Saibil, H. & Tsaneva, I.R. (2007). Dodecameric structure and ATPase activity of the human TIP48/TIP49 complex. J Mol Biol 366, 179–192.Google Scholar
Rachel, R., Jakubowski, U., Tietz, H., Hegerl, R. & Baumeister, W. (1986). Projected structure of the surface of Deinococcus radiodurans determined to 8Å resolution by cryomicroscopy. Ultramicroscopy 20, 305–316.Google Scholar
Raunser, S., Appel, M., Ganea, C., Geldmacher-Kaufer, U., Fendler, K. & Kühlbrandt, W. (2006). Structure and function of prokaryotic glutamate transporters from Escherichia coli and Pyrococcus horikoshii. Biochemistry 45, 12796–12805.Google Scholar
Ren, G., Gao, K., Bushman, F.D. & Yaeger, M. (2007). Single-particle image reconstruction of a tetramer of HIV integrase bound to DNA. J Mol Biol 366, 286–294.Google Scholar
Renault, L., Chou, H.T., Chiu, P.L., Hill, R.M., Zeng, X., Gipson, B., Zhang, Z.Y., Cheng, A., Unger, Y. & Stahlberg, H. (2006). Milestones in electron crystallography. Comput Aided Mol Design 20, 519–527.Google Scholar
Ruch, C., Skiniotis, G., Steinmetz, M.O., Walz, T. & Ballmer-Hofer, K. (2007). Structure of a VEGF-VEGF receptor complex determined by electron microscopy. Nat Struct Mol Biol 14, 249–250.Google Scholar
Schmidt-Krey, I., Haase, W., Mutucumarana, V., Stafford, D.W. & Kühlbrandt, W. (2007). Two-dimensional crystallization of human vitamin K-dependent γ–glutamyl carboxylase. J Struct Biol 157, 437–442.Google Scholar
Timasheff, S.N. (1998). Control of protein stability and reactions by weakly interacting cosolvents: The simplicity of the complicated. Adv Protein Chem 51, 255–432.Google Scholar
Toyoshima, C. & Unwin, N. (1988). Contrast transfer for frozen-hydrated specimens: Determination from pairs of defocused images. Ultramicroscopy 25, 279–291.Google Scholar
Tracz, E., Dickson, D.W., Hainfeld, J.F. & Ksiezak-Reding, H. (1997). Paired helical filaments in corticobasal degeneration: The fine fibrillary structure with NanoVan. Brain Res 773, 33–44.Google Scholar
Unwin, P.N.T. (1972). Negative staining of biological specimens using mixture salts. In Proceedings of the Fifth European Congress of the Electron Microscopy, pp. 232–233. London: Institute of Physics.
Unwin, P.N.T. (1975). Beef liver catalase structure: Interpretation of electron micrographs. J Mol Biol 98, 235–242.Google Scholar
Van Rooyen, J.M., Abratt, V.R. & Sewell, B.T. (2006). Three-dimensional structure of a type III glutamine synthetase by single-particle reconstruction. J Mol Biol 361, 796–810.Google Scholar
Viadiu, H., Gonen, T. & Walz, T. (2007). Projection map of aquaporin-9 at 7 Å resolution. J Mol Biol 367, 80–88.Google Scholar
Vinothkumar, K.R., Smits, S.H.J. & Kühlbrandt, W. (2005). pH-induced structural change in a sodium/proton antiporter from Methanococcus jannaschi. EMBO J 24, 2720–2729.Google Scholar
Wrigley, N.G. (1968). The lattice spacing of crystalline catalase as an internal standard of length in electron microscopy. J Ultrastruct Res 24, 454–464.Google Scholar