Summary
This method aims at providing structural information on protein or nucleoprotein complexes by high-resolution electron microscopy. The objective is to promote the self-assembly of the macromolecules into two-dimensional crystals in order to use electron crystallography methods. When combined with observations in the frozen hydrated states and dedicated image processing software these methods can provide detailed 3-D models of the complex. The 2-D crystals of soluble nucleoprotein complexes are formed on lipid monolayers spread at the air–water interface. The macromolecule of interest is targeted to the monolayer by either electrostatic or ligand-induced interactions with the hydrophilic head group of the lipid. Upon interaction with the lipids, the nucleoprotein complex is concentrated at the vicinity of the lipid layer whose in-plane mobility facilitates the contacts between macromolecules and the formation of ordered arrays.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Henderson, R., Baldwin, J.M., Ceska, T.A., Zemlin, F., Beckmann, E., and Downing, K.H. (1990). Model for the structure of bacteriorhodopsin based on high-resolution electron cryo-microscopy. J. Mol. Biol. 213, 899–929.
Kuhlbrandt, W., Wang, D.N., and Fujiyoshi, Y. (1994). Atomic model of plant light-harvesting complex by electron crystallography. Nature. 367, 614–621.
Amos, L.A., Henderson, R., and Unwin, P.N. Three-dimensional structure determination by electron microscopy of two-dimensional crystals. Prog. Biophys. Mol. Biol.39, 183–231.
van Heel, M., Gowen, B., Matadeen, R., Orlova, E.V., Finn, R., Pape, T., Cohen, D., Stark, H., Schmidt, R., Schatz, M., and Patwardhan, A. (2000). Single-particle electron cryo-microscopy: towards atomic resolution. Q. Rev. Biophys. 33, 307–369.
Frank, J. (2002). Single-particle imaging of macromolecules by cryo-electron microscopy. Annu. Rev. Biophys. Biomol. Struct. 31, 303–319.
Uzgiris, E.E., and Kornberg, R.D. (1983). Two-dimensional crystallization technique for imaging macromolecules, with application to antigen–antibody–complement complexes. Nature. 301, 125–129.
Schultz, P., Celia, H., Riva, M., Darst, S.A., Colin, P., Kornberg, R.D., Sentenac, A., and Oudet, P. (1990). Structural study of the yeast RNA polymerase A. Electron microscopy of lipid-bound molecules and two-dimensional crystals. J. Mol. Biol. 216, 353–362.
Darst, S.A., Ribi, H.O., Pierce, D.W., and Kornberg, R.D. (1988). Two-dimensional crystals of Escherichia coli RNA polymerase holoenzyme on positively charged lipid layers. J. Mol. Biol. 203, 269–273.
Lebeau, L., Regnier, E., Schultz, P., Wang, J.C., Mioskowski, C., and Oudet, P. (1990). Two-dimensional crystallization of DNA gyrase B subunit on specifically designed lipid monolayers. FEBS Lett. 267, 38–42.
Ribi, H.O., Reichard, P., and Kornberg, R.D. (1987) Two-dimensional crystals of enzyme-effector complexes: ribonucleotide reductase at 18-A resolution. Biochemistry. 26, 7974–7979.
Avila-Sakar, A.J., and Chiu, W. (1996). Visualization of beta-sheets and side-chain clusters in two-dimensional periodic arrays of streptavidin on phospholipid monolayers by electron crystallography [published erratum appears in Biophys. J.71, 517]. Biophys. J. 70, 57–68.
Lebeau, L., Schultz, P., Célia, H., Mésini, P., Nuss, S., Klinger, C., Olland, S., Oudet, P., and Nioskowski, C. (1996). Specifically designed lipid assemblies as tools for two-dimensional crystallization of soluble macromolecules, in Handbook of Nonmedical Applications of Liposomes 11 (Barenllolz, Y. and Lasic, D.D., eds.) CRC Press, Boca Raton, pp. 133–186.
Darst, S.A., Ahlers, M., Meller, P.H., Kubalek, E.W., Blankenburg, R., Ribi, H.O., Ringsdrof, H., and Kornberg, R.D. (1991). Two-dimensional crystals of streptavidin on biotinylated lipid layers and their interactions with biotinylated macromolecules. Biophys. J. 59, 387–396.
Celia, H., Jontes, J.D., Whittaker, M., and Milligan, R.A. (1996). Two-dimensional crystallization of brush border myosin I. J. Struct. Biol. 117, 236–241.
Venien-Bryan, C., Balavoine, F., Toussaint, B., Mioskowski, C., Hewat, E.A., Helme, B., and Vignais, P.M. (1997). Structural study of the response regulator HupR from rhodobacter capsulatus. Electron microscopy of two-dimensional crystals on a nickel-chelating lipid. J. Mol. Biol. 274, 687–692.
Kubalek, E.W., Le Grice, S.F., and Brown, P.O. (1994). Two-dimensional crystallization of histidine-tagged, HIV-1 reverse transcriptase promoted by a novel nickel-chelating lipid. J. Struct. Biol. 113, 117–123.
Bischler, N., Balavoine, F., Milkereit, P., Tschochner, H., Mioskowski, C., and Schultz, P. (1998). Specific interaction and two-dimensional crystallization of histidine tagged yeast RNA polymerase I on nickel-chelating lipids. Biophys. J. 74, 1522–1532.
Mosser, G., and Brisson, A. (1991). Structural analysis of two-dimensional arrays of cholera toxin B-subunit. J. Electron Microsc. Tech. 18, 387–394.
Crucifix, C., Uhring, M., and Schultz, P. (2004). Immobilization of biotinylated DNA on 2-D streptavidin crystals. J. Struct. Biol. 146, 441–451.
Kubalek, E.W., Kornberg, R.D., and Darst, S.A. (1991). Improved transfer of two-dimensional crystals from the air/water interface to specimen support grids for high-resolution analysis by electron microscopy. Ultramicroscopy. 35, 295–304.
Fukami, A., and Adachi, K. (1965). A new method of preparation of a self-perforated micro plastic grid and its application. J. Electron Microsc. 14, 112–118.
Dubochet, J., Adrian, M., Chang, J.J., Homo, J.C., Lepault, J., McDowall, A.W., and Schultz, P. (1988). Cryo-electron microscopy of vitrified specimens. Q. Rev. Biophys. 21, 129–228.
Asturias, F.J., and Kornberg, R.D. (1995). A novel method for transfer of two-dimensional crystals from the air/water interface to specimen grids. EM sample preparation/lipid-layer crystallization. J. Struct. Biol. 114, 60–66.
Schmutz, M., and Brisson, A. (1996). Analysis of carbon film planarity by reflected light microscopy. Ultramicroscopy. 63, 263–272.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Schultz, P., Crucifix, C., Lebeau, L. (2009). Two-Dimensional Crystallisation of Soluble Protein Complexes. In: Leblanc, B., Moss, T. (eds) DNA-Protein Interactions. Methods in Molecular Biology™, vol 543. Humana Press. https://doi.org/10.1007/978-1-60327-015-1_22
Download citation
DOI: https://doi.org/10.1007/978-1-60327-015-1_22
Published:
Publisher Name: Humana Press
Print ISBN: 978-1-60327-014-4
Online ISBN: 978-1-60327-015-1
eBook Packages: Springer Protocols