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The potential and limitations of neutrons, electrons and X-rays for atomic resolution microscopy of unstained biological molecules

Published online by Cambridge University Press:  17 March 2009

Richard Henderson
Richard Henderson
Affiliation:
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, UK
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Summary

Radiation damage is the main problem which prevents the determination of the structure of a single biological macromolecule at atomic resolution using any kind of microscopy. This is true whether neutrons, electrons or X-rays are used as the illumination. For neutrons, the cross-section for nuclear capture and the associatedenergy deposition and radiation damage could be reduced by using samples that are fully deuterated and 15N-labelled and by using fast neutrons, but single molecule biological microscopy is still not feasible. For naturally occurring biological material, electrons at present provide the most information for a given amount of radiation damage. Using phase contrast electron microscopy on biological molecules and macromolecular assemblies of ˜ 105 molecular weight and above, there is in theory enough information present in the image to allow determination of the position and orientation of individual particles: the application of averaging methods can then be used to provide an atomic resolution structure. The images of approximately 10000 particles are required. Below 105 molecular weight, some kind of crystal or other geometrically ordered aggregate is necessary to provide a sufficiently high combined molecular weight to allow for the alignment. In practice, the present quality of the best images still falls short of that attainable in theory and this means that a greater number of particles must be averaged and that the molecular weight limitation is somewhat larger than the predicted limit. For X-rays, the amount of damage per useful elastic scattering event is several hundred times greater than for electrons at all wavelengths and energies and therefore the requirements on specimen size and number of particles are correspondingly larger. Because of the lack of sufficiently bright neutron sources in the foreseeable future, electron microscopy in practice provides the greatest potential for immediate progress.

Type
Research Article
Information
Quarterly Reviews of Biophysics , Volume 28 , Issue 2 , May 1995 , pp. 171 - 193
Copyright
Copyright © Cambridge University Press 1995

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References

Abramowitz, M. & Stegun, I. A. (1965). Handbook of Mathematical Functions with Formulas, Graphs and Mathematical Tables. New York: Dover Publications.Google Scholar
Adrian, M., Heggeler-Bordier, B. T., Wahli, W., Stasiak, A. Z., Stasiak, A. & Dubochet, J. (1990). Direct visualisation of supercoiled DNA molecules in solution. EMBO J. 9, 4551–4554.CrossRefGoogle ScholarPubMed
Arndt, U. (1984). Optimum X-ray wavelength for protein crystallography. J. Appl. Cryst. 17, 118–119.CrossRefGoogle Scholar
Baker, T. S., Newcomb, W. W., Olson, N. H., Cowsert, L. M., Olson, C. & Brown, J. D. C. (1991). Structures of bovine and human papillomaviruses – analysis by cryoelectron microscopy and 3-dimensional image-reconstruction. Biophys. J. 60, 1445–1456.CrossRefGoogle Scholar
Breedlove, J. R. & Trammell, G. T. (1970). Molecular microscopy: fundamental limitations. Science, 170, 1310–1313.CrossRefGoogle ScholarPubMed
Cowley, J. M. (1975). Diffraction Physics Amsterdam: North-Holland.Google Scholar
Darwin, C. G. (1914). The theory of X-ray reflexion. Phil. Mag. 27, 315–333.CrossRefGoogle Scholar
De Rosier, D. J. & Klug, A. (1968). Reconstruction of three-dimensional structures from electron micrographs. Nature 217, 130–134.CrossRefGoogle ScholarPubMed
Erickson, H. & Klug, A. (1971). Measurement and compensation of defocusing and aberrations by Fourier processing of electron micrographs. Phil. Trans. Roy. Soc. Lond., B 261, 105–118.Google Scholar
Gabor, D. (1948). A new microscopic principle. Nature, 161, 777–778.CrossRefGoogle ScholarPubMed
Glaeser, R. M. & Ceska, T. A. (1989). High-voltage electron diffraction from bacteriorhodopsin (purple membrane) is measurably dynamical. Acta Cryst. A45, 620–628.CrossRefGoogle Scholar
Gonzalez, A., Thompson, A. W. & NAVE, C. (1992). Cryo-protection of protein crystals in intense X-ray beams. Rev. Sci. Instrum. 63, 1177–1180.CrossRefGoogle Scholar
Henderson, R. (1990). Cryo-protection of protein crystals against radiation damage in electron and X-ray diffraction. Proc. Roy. Soc. B 241, 6–8.Google Scholar
Henderson, R. (1992). Image contrast in high resolution electron microscopy of biological macromolecules: TMC in ice. Ultramicroscopy 46, 1–18.CrossRefGoogle Scholar
Henderson, R. & Glaeser, R. M. (1985). Quantitative analysis of image contrast in electron micrographs of beam-sensitive crystals. Ultramicroscopy 16, 139–150.CrossRefGoogle Scholar
Henderson, R., Baldwin, J. M., Ceska, T. A., Beckmann, E., Zemlin, F. & Downing, K. (1990). A model for the structure of bacteriorhodopsin based on high resolution electron cryomicroscopy. J. Mol. Biol. 213, 899–929.CrossRefGoogle Scholar
Hoppe, W. (1983). Electron diffraction with the transmission electron microscope as a phase-determining diffractometer - from spatial frequency filtering to the three-dimensional structure analysis of ribosomes. Angew. Chem. Int. Ed. Engl. 22, 456–485.CrossRefGoogle Scholar
Hubbell, J. H., Gimm, H. A. & Øverbø, I. (1980). Pair, triplet and total atomic cross sections (and mass attenuation coefficients) for 1Mev-100Gev photons in elements Z = 1 to 100. J. Phys. Chem. Ref. Data 9, 1023–1147.CrossRefGoogle Scholar
Jacobsen, C., Williams, S., Anderson, E., Browne, M. T., Buckley, C. J., Kern, D., Kirz, J., Rivers, M. & Zhang, X. (1991). Diffraction-limited imaging in a scanning transmission X-ray microscope. Optics Commun. 86, 351–364.CrossRefGoogle Scholar
Jap, B. K., Walian, P. J. & Gehring, K. (1991). Structural architecture of an outermembrane channel as determined by electron crystallography. Nature 350, 167–170.CrossRefGoogle Scholar
Jeng, T.-W., Crowther, R. A., Stubbs, G. & Chiu, W. (1989). Visualisation of alphahelices in tobacco mosaic virus by cryo-electron microscopy. J. Mol. Biol. 205, 251–257.CrossRefGoogle ScholarPubMed
Kirz, J., Ade, H., Jacobsen, C., Ko, C.-H., Lindaas, S., McNulty, I., Sayre, D., Williams, S. & Zhang, X. (1992). Soft X-ray microscopy with coherent X-rays. Rev. Set. Instrum. 63, 557–563.CrossRefGoogle Scholar
Kühlbrandt, W. & Wang, D. N. (1991). Three-dimensional structure of plant lightharvesting complex determined by electron crystallography. Nature 350, 130–134.CrossRefGoogle ScholarPubMed
Kühlbrandt, W., Wang, D. N. & Fujiyoshi, Y. (1994). Atomic model of plant lightharvesting complex by electron crystallography. Nature 367, 614–621.CrossRefGoogle ScholarPubMed
Langmore, J. P. & Smith, M. F. (1992). Quantitative energy-filtered electron microscopy of biological molecules in ice. Ultra-microscopy 46, 349–373.Google ScholarPubMed
Licht, H. (1991). Optimum focus for taking electron holograms. Ultramicroscopy 38, 13–22.CrossRefGoogle Scholar
Penczek, P., Radermacher, M. & Frank, J. (1992). Three-dimensional reconstruction of single particles embedded in ice. Ultramicroscopy 40, 33–53.CrossRefGoogle ScholarPubMed
Reimer, L. (1989). Transmission Electron Microscopy, 2nd ed. Berlin: Springer-Verlag.CrossRefGoogle Scholar
Sayre, D. & Chapman, H. N. (1994). X-ray microscopy. Acta Cryst. A51 (In press).Google Scholar
Sayre, D., Kirz, J., Feder, R., Kim, D. M. & Spiller, E. (1977). Transmission microscopy of unmodified biological materials: comparative radiation dosages with electrons and ultrasoft X-ray photons. Ultramicroscopy 2, 337–349.CrossRefGoogle ScholarPubMed
Sears, V. F. (1992). Neutron scattering lengths and cross-sections. Neutron News 3(3), 26–37.CrossRefGoogle Scholar
Smith, M. F. & Langmore, J. P. (1992). Quantitation of molecular densities in cryoelectron microscopy: determination of the radial density distribution of tobacco mosaic virus. J. Mol. Biol. 226, 763–774.CrossRefGoogle ScholarPubMed
Spence, J. C. H. (1988). Experimental High-Resolution Electron Microscopy, 2nd ed., Oxford University Press.Google Scholar
Stewart, P. L., Burnett, R. M., Cyrklaff, M. & Fuller, S. D. (1991). Image reconstruction reveals the complex molecular organisation of Adenovirus. Cell 67, 145–154.CrossRefGoogle ScholarPubMed
Steyerl, A. (1989). Ultracold neutrons: production and experiments. Physica B, 156, 528–533.CrossRefGoogle Scholar
Steyerl, A., Drexel, W., Ebisawa, T., Gutsmiedle, E., Steinhauser, K.-A., Gahler, R., Mampe, W. & Ageron, P. (1988). Neutron microscopy. Rev. Phys. Appl. 23, 171–180.CrossRefGoogle Scholar
Thon, F. (1966). Zur Defokussierungsabhängigkeit des Phasenkontrastes bei der elektronenmikroskopischen Abbildung. Z. Naturforsh. 219, 476–478.CrossRefGoogle Scholar
Tonomura, A. (1992). Electron-holographic interference microscopy. Adv. Physics 41, 50–103.CrossRefGoogle Scholar
Toyoshima, C., Sasabe, H. & Stokes, D. L. (1993). Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane. Nature 362, 469–471.CrossRefGoogle ScholarPubMed
Toyoshima, C. & Unwin, N. (1988a). Contrast transfer for frozen-hydrated specimens: determination from pairs of defocused images. Ultramicroscopy 25, 279–292.CrossRefGoogle ScholarPubMed
Toyoshima, C. & Unwin, N. (1988b). Ion channel of acetyl-choline receptor reconstructed from images of postsynaptic membrane. Nature 336, 247–250.CrossRefGoogle Scholar
Zernike, F. (1955). How I discovered phase contrast. Science 121, 345–349.CrossRefGoogle Scholar