Practical Protein Chemistry - A. Darbre 1989

X-ray crystallography and electron microscopy
X-ray diffraction
Imaging of molecules: a type of structural analysis

When examining a specimen under a conventional Light Microscope, the light scattered by the object is focused by the objective lens, thereby forming a magnified image (Fig. 20.1). As light waves are scattered by the specimen, they undergo phase changes, and the objective lenses, upon focusing them, preserve the post-scattering phase relationships. Consequently, when the rays recombine, an image of the object is formed (Fig. 20.2). Resolution, i.e., the minimum size of details reproducible in such an image, is limited by the wavelength of the light used (typically ∼600 nm). Finer details could potentially be observed using a microscope operating, for example, on X-rays with a wavelength of 0.154 nm (CuKα-line). Unfortunately, lenses capable of focusing X-rays have not yet been developed, making image formation under such conditions impossible. One can only record the intensities of the rays scattered by the object under study, for instance, using a photographic plate. However, the phases of these rays remain unknown, making it impossible to reconstruct the image of the object.

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FIG. 20.1. Schematic diagram of a light microscope with Glass lenses that collect the light scattered by the specimen and reconstruct a magnified image of the object. This design makes it possible to preserve the Phases of the scattered rays in their original relationship.

The object considered thus far could consist of many molecules in various orientations, such as protein molecules in solution. An image of such a specimen would reproduce each molecule in its specific orientation. However, by collecting data solely on ray intensities, it is impossible to isolate the rays scattered by a particular molecule from those scattered by surrounding solvent molecules. This can be achieved by using crystalline samples, where all molecules adopt the same orientation (Fig. 20.3). Due to the regularity of the crystal, the rays scattered by these molecules are concentrated into distinct diffraction peaks, making it possible to separate the radiation scattered by the protein molecules from Background scattering caused, for example, by solvent molecules, thereby significantly increasing the signal-to-noise ratio. Studying crystalline samples can only yield an averaged image of the molecule of the substance under investigation within the crystal lattice, rather than an individual image of a specific molecule freely floating in solution. The diffraction pattern from a crystal (see, e.g., Fig. 20.4) can be recorded using a suitably designed X-ray camera. The diffraction pattern reflects the three-dimensional Structure OF THE crystal under study. Therefore, a complete set of scattered ray intensity data must include a series of such diffraction patterns, stacked one above another to form the so-called three-dimensional reciprocal lattice. The distances between the nodes of this lattice are inversely proportional to the intermolecular distances in the crystal. The intensities of the diffraction maxima, or spots, contain information about the Fine Structure of the molecule. If one could determine the relative phase values of the waves upon intersecting the photographic plate, it would be possible to calculate the Molecular structure and thereby obtain its image. However, determining the phases—or solving the so-called phase problem—remains one of the most laborious stages in X-ray structure analysis.

FIG. 20.2. Two light waves (a and b) with different amplitudes and phases and their sum (c). It can be seen that both amplitude and phase information are required for summation.