X-ray diffraction The spacing of atoms in a crystal lattice can be determined by measuring the locations and intensities of points created on photographic film by an X-ray beam of a particular wavelength after the beam has been diffracted. by the electrons of the atoms. For example, X-ray analysis of sodium chloride crystals shows that the Na and Cl ions are arranged in a simple cubic lattice. The distances between different types of atoms in complex organic molecules, including very large ones such as proteins, can also be analyzed using X-ray diffraction methods. However, the technique for analyzing crystals of complex molecules is much more complex than that of simple salt crystals. If the repeating pattern of the crystal is a protein-sized molecule, for example, the numerous atoms in the molecule result in thousands of diffraction points that need to be analyzed by computer. The process can be understood at an elementary level by considering how images are created in an optical microscope. Light from a point source is focused on an object. Light waves are scattered by the object and these scattered waves are recombined by a series of lenses to create a magnified image of the object. The smallest object whose structure can be determined with such a system, i.e. H. the resolution of the microscope is determined by the computer.
Wavelengths in the range of 400 to 700 nm Objects that are less than half the wavelength of the incident light cannot be resolved. To solve objects as small as proteins, we need to use X-rays with wavelengths in the range of 0.7 to 1.5 Å (0.07 to 0.15 nm). However, there are no lenses that can recombine X-rays into an image; instead, the pattern of the diffracted X-rays is collected directly and an image is reconstructed using mathematical techniques. The information content of X-ray crystallography depends on the degree of structural order of the sample. Some important structural parameters were obtained from the first studies of the diffraction patterns of fiber proteins, which are arranged in fairly regular arrangements in hair and wool. However, the ordered bundles made up of fiber proteins are not crystals, the molecules are lined up side by side, but not all are lined up in the same direction. The most detailed three-dimensional structural information of proteins requires a highly ordered protein crystal. Protein crystallization is an empirical science and the structures of many important proteins are not yet known simply because they have proven difficult to crystallize. Practitioners have compared making protein crystals to holding a stack of bowling balls together with cellophane tape. X-ray structure analysis is performed surgically in several steps. Once a crystal is obtained, it is placed in an X-ray beam between the X-ray source and a detector and a regular array of spots called reflection is generated. The spots are created by the diffracted x-ray beam, and each atom in a molecule makes a contribution to each spot. An electron-density map of the protein is reconstructed from the overall diffraction pattern of spots by using a mathematical technique called a Fourier transform. In effect, the computer acts as a “computational lens.” A model for the structure is then built that is consistent with the electron-density map.