Protein molecules are very small 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 many (~ one trillion) 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 interference of waves as they pass through the protein crystal and are scattered in many directions. The brightness of the diffracted X-rays gives us the information we need to 'see' individual protein molecules.

We produce X-rays in the laboratory using a X-ray generator. These machines use a filament, similar to that in a light bulb, which fires electrons into a piece of metal called a rotating anode. As the electrons hit the metal, X-rays are produced. The X-ray beam is directed at the crystal, 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.

Diffraction from protein crystals can be very weak, and we usually need to use more intense sources, called synchrotrons, as well. Synchrotrons provide from 1-100 million 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.