Electron crystallography

Electron crystallography is a method to determine the arrangement of atoms in solids using an electron microscope. It can complement X-ray crystallography for studies of very small crystals (<0.1 micrometers), both inorganic, organic and proteins, such as membrane proteins, that cannot easily form the large 3-dimensional crystals required for that process. Protein structures are usually determined from either 2-dimensional crystals (sheets or helices), polyhedrons such as viral capsids, or dispersed individual proteins. Electrons can be used in these situations, whereas X-rays cannot, because electrons interact more strongly with atoms than X-rays do. Thus, X-rays will travel through a thin 2-dimensional crystal without diffracting significantly, whereas electrons can be used to form an image. Conversely, the strong interaction between electrons and proteins makes thick (e.g. 3-dimensional > 1 micrometer) crystals impervious to electrons, which only penetrate short distances.

One of the main difficulties in X-ray crystallography is determining phases in the diffraction pattern. Because no X-ray lens exists, X-rays cannot be used to form an image of the crystal being diffracted, and hence phase information is lost. Fortunately, electron microscopes contain electron lenses, and so the crystallographic structure factor phase information can be experimentally determined in electron crystallography. Aaron Klug was the first to realise that the phase information could be read out directly from the Fourier transform of an electron microscopy image that had been scanned into a computer, already in 1968. For this, and his studies on virus structures and transfer-RNA, Klug received the Nobel Prize for chemistry in 1982.

A common problem to X-ray crystallography and electron crystallography is radiation damage, by which especially organic molecules and proteins are damaged as they are being imaged, limiting the resolution that can be obtained. This is especially troublesome in the setting of electron crystallography, where that radiation damage is focused on far fewer atoms. One technique used to limit radiation damage is electron cryomicroscopy, in which the samples undergo cryofixation and imaging takes place at liquid nitrogen or even liquid helium temperatures. Because of this problem, X-ray crystallography has been much more successful in determining the structure of proteins that are especially vulnerable to radiation damage.

The first electron crystallographic protein structure to achieve atomic resolution was bacteriorhodopsin, determined by Richard Henderson and coworkers at the Medical Research Council Laboratory of Molecular Biology in 1990. But already in 1975 Unwin and Henderson had determined the first membrane protein structure at intermediate resolution (7 Ångström), showing for the first time the internal structure of a membrane protein, with its alpha-helices standing perpendicular to the plane of the membrane. Since then, several other high-resolution structures have been determined by electron crystallography, including the light-harvesting complex, the nicotinic acetylcholine receptor, and the bacterial flagellum^{[citation needed]}.

Electron microscopy image of an inorganic tantalum oxide, with its Fourier transform, also called diffractogram, inset. Notice how the appearance changes from the upper thin region to the thicker lower region. The unit cell of this compound is about 15 by 25 Ångström. It is outlined at the centre of the figure, inside the result from image processing, where the symmetry has been taken into account. The black dots show clearly all the tantalum atoms. The diffraction extends to 6 orders along the 15 Å direction and 10 orders in the perpendicular direction. Thus the resolution of the EM image is 2.5 Å (15/6 or 25/10). This calculated Fourier transform contain both amplitudes (as seen) and phases (not displayed).

Electron diffraction pattern of the same crystal of inorganic tantalum oxide shown above. Notice that there are many more diffraction spots here than in the diffractogram calculated from the EM image above. The diffraction extends to 12 orders along the 15 Å direction and 20 orders in the perpendicular direction. Thus the resolution of the ED pattern is 1.25 Å (15/12 or 25/20). ED patterns do not contain phase information, but the clear differences between intensities of the diffraction spots can be used in crystal structure determination.

Electron crystallographic studies of inorganic crystals were first done by Aaron Klug in 1978 and Hovmöller and coworkers in 1984. At first, only electron microscopy (EM) images were used, because with those it is possible to select only the very thin edge of the crystal. In the thicker parts of an inorganic crystal (> 20 nanometers or so) the electrons scatter multiple times, making the interpretation of the image very hard or even impossible. Not only do the EM images change with crystal thickness, they are also very sensitive to slight changes of focus. Unlike our daily experience with photographic cameras, EM images may look sharp within a range of focus values. Thus image simulations were developed (by Michael O'Keefe) as a way to show how EM images change their appearance with variations of thickness and focus.

A very unfortunate and confusing scientific discussion has ravaged the field of electron microscopy of inorganic compounds; while some have claimed that "the phase information is present in EM images" others have the opposite view that "the phase information is lost in EM images". The reason for these opposite views is that the word "phase" has been used with different meanings in the two communities of physicists and crystallographers. The physicists are more concerned about the "electron wave phase" - the phase of a wave moving through the sample during exposure by the electrons. This wave has a wavelength of about 0.02-0.03 Ångström (depending on the accelerating voltage of the electron microscope). Its phase is related to the phase of the undiffracted direct electron beam. The crystallographers, on the other hand, mean the "crystallographic structure factor phase" when they simply say "phase". This phase is the phase of standing waves of potential in the crystal (very similar to the electron density measured in X-ray crystallography). Each of these waves have their specific wavelength, called d-value for distance between so-called Bragg planes of low/high potential. These d-values range from the unit cell dimensions to the resolution limit of the electron microscope, i.e. typically from 10 or 20 Ångström down to 1 or 2 Ångström. Their phases are related to a fixed point in the crystal, defined in relation to the symmetry elements of that crystal. The crystallographic phases are a property of the crystal, so they exist also outside the electron microscope. The electron waves vanish if the microscope is switched off. In order to determine a crystal structure, it is necessary to know the crystallographic structure factors, but not to know the electron wave phases.

Just as with proteins, it has been possible to determine the atomic structures of inorganic crystals by electron crystallography. For simpler structure it is sufficient to use three perpendicular views, but for more complicated structures, also projections down ten or more different diagonals may be needed.

In addition to electron microscopy images, it is also possible to use electron diffraction (ED) patterns. The utmost care must be taken to record the ED patterns from thin areas. Just as with X-ray diffraction patterns, the important crystallographic structure factor phase is lost in electron diffraction patterns. On the other hand, ED patterns of inorganic crystals often have very high resolution, giving amplitude information higher than 1 Ångström. This is about twice the EM image resolution (2 Ångström) of the best standard electron microscopes.

Recently, two very complicated zeolite structures have been determined by electron crystallography combined with X-ray powder diffraction (Gramm and coworkers 2006, Baerlocher and coworkers 2007). These are more complex than the most complex zeolite structures determined by X-ray crystallography.

A review of where electron crystallography stands today was published by Zou and Hovmöller 2008.

References

• Hovmöller,S, Sjögren,A, Farrants,G, Sundberg,M & Marinder,B.O. (1984) Accurate Atomic Positions from Electron Microscopy. Nature vol 311, p. 238-241.

• K. H. Downing, H. Meisheng, H.-R. Wenk & M. A. O'Keefe (1990) Resolution of oxygen atoms in staurolite by three-dimensional transmission electron microscopy. Nature vol 348, p. 525 - 528.

• F.Gramm, C.Baerlocher, L.B.McCusker, S.J.Warrender, P.A.Wright, B.Han, S.B.Hong, Z.Liu, T.Ohsuna, & O.Terasaki (2006).Complex zeolite structure solved by combining powder diffraction and electron microscopy Nature, Vol. 444, p. 79-81.

• C. Baerlocher, F. Gramm, L. Massüger, L. B. McCusker, Z.B. He, S. Hovmöller, X.D. Zou (2007) Structure of the Polycrystalline Zeolite Catalyst IM-5 Solved by Enhanced Charge Flipping.

Science Vol. 315 p 1113 - 1116.

• Zou, X.D. and Hovmöller, S. (2008) Electron crystallography: Imaging and Single Crystal Diffraction from Powders. Acta Cryst vol. A 64 p. 149-160.

External links

• Interview with Aaron Klug Nobel Laureate for work on crystallograph electron microscopy Freeview video by the Vega Science Trust.

• Raunser S. & Walz T. (2009). "Electron Crystallography as a Technique to Study the Structure on Membrane Proteins in a Lipidic Environment". Annual Review of Biophysics 38 (1). doi:10.1146/annurev.biophys.050708.133649. http://arjournals.annualreviews.org/doi/abs/10.1146/annurev.biophys.050708.133649.