Max von Laue demonstrated that X-rays are electromagnetic waves, having the same nature as visible light or radio waves. The only difference is the very short wavelength of X-rays, which is around 1-1.5 Å (1 Ångström is 10-10
meters). For comparison, the wavelength of visible light is between 400 to 700 nm (one nm is 10 Å).
X-ray diffraction is caused by the interaction of electromagnetic waves with the atoms inside the crystals, particularly with the electrons. The waves get scattered by the electrons; each electron becomes a miniature X-ray source. Scattered waves from all the electrons within each atom are added to each other, giving diffracted waves from each atom, etc. When the scattered waves are added, they may either get stronger or cancel each other out (in optics, this process is called interference). As shown in the figure below, the X-ray detector registers those that get stronger. Interestingly, we do not necessarily need X-rays to observe interference; we can, for example, go to a lake nearby, through two stones into the water, and then watch how the waves from the two rocks either reinforce each other or become weaker. There are many demonstrations of wave addition on the web; one can be found here
.X-ray data collection, electron density calculation, and model building
X-rays may be generated using various laboratory X-ray sources or at synchrotrons, where very high intensity and highly focused X-rays can be generated. Several synchrotrons worldwide have stations adapted for collecting X-ray data from protein crystals. Depending on the type of crystal (crystals may have different cell dimensions and symmetry groups), different strategies for data collection are followed. Usually, the crystal is rotated in the X-ray beam one degree at a time and exposed to X-rays for a short period (seconds or even less) until a complete data set is collected. The total data collection time depends on the intensity of the X-ray source, the size of the crystal, and how well it diffracts. The data are subsequently processed using specific software (a process in crystallography called "data reduction").
Each spot on the image below is a diffracted X-ray beam, which emerged from the crystal and was registered by the X-ray detector. Thousands of diffraction spots must be collected from a protein crystal to get a complete data set. The intensities of the diffraction spots are extracted during data processing and used to calculate an electron density map
of the molecules inside the crystal. The electron density, in turn, will tell us where the atoms are located, information that is used to build a model of the molecule. The image on the right shows a side chain of tryptophan built into its electron density.