The difficulty in applying X-ray diffraction techniques to molecules made up of thousands of atoms — macromolecules — stems from the increasing number of diffracted and reflected rays. Not only the amplitudes of all of these rays need to be measured to construct a useful image, but also the phase, a technical challenge often referred to as the 'phase problem'. Multiple-wavelength anomalous diffraction (MAD) reduces the problem from one with many different types of atom to one that involves just one or two that scatter X-rays in an unusual way. The information gained in this way serves as a reference that provides an insight into the structure of the entire molecule.
Anomalous diffraction occurs when the energy of the incident photons is resonant with an electron transition in a metallic atom, leading to the scattering of light. Anomalous scattering differs from normal scattering in that, as it is based on a resonance, it is very wavelength-dependent (but almost independent of scattering angle). By probing the sample with different wavelength X-rays, wavelength-dependent and wavelength-independent elements of the diffraction pattern can be separated. This approach has proved a boon in structural biology, and can even be applied to macromolecules that have no metallic content, by introducing the appropriate heavy metal atoms.
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