Determining the structure of a molecule, especially a protein, is an important step in determining its function. In many diseases, changes in molecular structure are involved in the pathologic process, and understanding these changes can help in the design of therapy. The three-dimensional arrangement of atoms in a molecule can be determined using a variety of physical techniques. For large biological macromolecules the most common experimental techniques for structure determination include X-ray crystallography, electron microscopy, and nuclear magnetic resonance (NMR). Finally, approximate models depicting the three-dimensional arrangement of atoms can be built using computer modeling.
The principles behind the use of X rays for the determination of the structures of biological macromolecules are quite different from the use of "X rays" in the practice of medicine. A medical X-ray film shows a shadow revealing internal body parts depending on how easily the X rays penetrated them. In contrast, X-ray crystallography looks at how X rays are diffracted, or scattered, by the atoms in a sample and determines what the three-dimensional arrangement of the atoms must be to give rise to the observed pattern of scattering. (This is somewhat akin to determining the structure of a jungle gym by bouncing a tennis ball off it and recording the pattern of bounces.)
The distances between atoms in a molecule are very small, on the order of 10 –10 meters, and the wavelength of the radiation used to determine their relative positions must be correspondingly small. X rays have the necessary small wavelength. The amount of radiation scattered by one molecule is too small to measure; therefore, it is necessary to combine the diffraction from a large number of molecules. Crystals are used because they contain an ordered arrangement of many molecules. Computers are used to reconstruct an image of the molecules in the crystal. The technique of X-ray crystallography provides the most detailed and accurate information on the structure of biological macromolecules.
Electron microscopy uses an electron beam to study the structure of biological materials. By using a magnet to focus electrons scattered from a sample, an electron microscope can form an image in a manner similar to a conventional microscope. One problem is that the electron beam used in such a microscope has a very high energy and can destroy sensitive biological samples. To aid in its preservation, the sample is often maintained at a very low temperature (this is called cryo-electron microscopy). As in the case of X-ray diffraction, it is an advantage to combine the electrons scattered from many molecules to get an average image. This can be done using ordered samples such as two-dimensional crystals or by orienting and averaging many images. Electron microscopy is especially useful for large complexes of macromolecules.
Nuclear magnetic resonance is not a scattering technique, but a spectroscopic technique that depends on the interaction of atomic nuclei with radio-frequency radiation and a magnetic field. This interaction is very sensitive to the environment surrounding an atom, and therefore can be used to determine what other atoms are nearby a given atom. Once the features in an NMR spectrum have been associated with specific atoms it is possible to combine this experimental information of the local arrangement of atoms with knowledge of the chemical structure of a molecule to derive a three-dimensional structure. An advantage of NMR is that it examines molecules in solution and can also provide information about their dynamic properties, or motion.
In addition to the experimental techniques for determining the three-dimensional structures of molecules, it is possible to use computational techniques to predict structures. The most successful approach to structure prediction utilizes the observation that proteins with similar amino acid sequences have similar three-dimensional structures. This allows one to predict an approximate structure if a structure of a related protein is already known. This starting point can then be combined with knowledge of the chemical structure and physical principles to improve the model.
Wayne F. Anderson
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Branden, Carl, and John Tooze. Introduction to Protein Structure, 2nd ed. New York: Garland Publishing, 1999.
Stryer, Lubert. Biochemistry, 4th ed. New York: W. H. Freeman and Company, 1995.