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Protein molecules are very small (about 10-6 mm in one dimension) and cannot be seen under a light microscope since the wavelength of light is larger than the protein size. Protein crystals are made up of an array of many (~1011-1012) identical molecules at regularly spaced intervals. The regular spacing allows a technique called diffraction to determine the structure of the protein making up a crystal. In very simple terms, diffraction is the bending, spreading, or interference of waves as they pass through an obstruction or gap. The gaps between protein molecules are small. Therefore, X-rays, with a wavelength of ~0.1 nm (10x10-7 mm), are diffracted by the array of proteins in the crystal and give us the information we need to 'see' individual protein molecules.
We generate X-rays in the laboratory using a machine called a rotating anode X-ray generator. These machines use a filament, similar to that in a light bulb, which fires electrons onto a spinning metal cylinder (the rotating anode). As the electrons hit the metal, X-rays are produced. The X-ray beam is directed at the crystalline sample, which is typically kept very cold (100°K or -278°F) using a jet of dry nitrogen gas. Cooling reduces radiation damage to the crystal during the experiment.
Only a tiny portion of the X-rays is diffracted; most of the radiation passes straight through the crystal and is stopped by metal shielding. The diffracted X-rays form a pattern of spots that extend outward from the center of the X-ray beam. In our laboratory, these spots are recorded on a phosphor screen in a device called an image plate. The positions and intensities of these spots are measured and used to determine the structure.
Although rotating anodes provide a bright source of X-rays, diffraction from protein crystals can be very weak, and we usually need to use even more intense sources, called synchrotrons, as well. Synchrotrons provide from 106-108 more X-rays than are available from our rotating anodes. Synchrotrons allow fast moving electrons in circular orbits to loose energy in the form of electromagnetic waves such as X-rays. Our laboratory rotating anode X-ray sources are small; you could fit one in a single parking space. On the other hand, synchrotrons can be hundreds of meters in diameter, and there are only a few in the US. We use the synchrotrons at Cornell and Stanford Universities and at the Brookhaven and Argonne National Laboratories. Part of our developing methodologies research seeks to use neutron radiation rather than X-rays to visualize atomic detail. Currently, we make use of neutron sources in Grenoble (France) and, in the not too distant future, will use neutrons from the new Spallation Neutron Source in Oak Ridge, Tennessee.
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