X-ray diffraction enables determination of the structure of proteins at near atomic resolution. The approach requires formation of a protein crystal, which contains solvent and is thus a concentrated solution, for use as the target. Our understanding of the detailed components of protein structurederived from this crystalline state correlate well with other physical measurements of protein in solution [e.g., by NMR spectroscopy (see p. 128)].
Generation of protein crystal can be the most time-consuming aspect of the process. Proteins have molecular dimensions at least an order of magnitude greater than a small molecule, and the packing of large protein molecules into the crystal latticegenerates a crystal with large “holes” or solvent channels. A protein crystal typically contains 40-60% solvent, and it may be considered a concentrated solution rather than the hard crystalline solid obtain with most small molecules. The presence of solvent and unoccupied volume in the crystal permits the infusion of inhibitors and substrates into the protein molecules in the “crystalline state.”
Adynamic flexibility within regions of the protein structure may be seen as “disorder” in the X-ray diffraction pattern. Disorder describes the situation in which the observed electron destiny can be fitted by more than a single local conformation. Two explanations must be distinguished. The first involves the presence of two or more static molecular conformations, which are present in astoichiometric relationship. The second involves the actual dynamic range of motion exhibited by atoms or groups of atoms in localized regions of the molecule. These explanations can be distinguished by lowering the temperature of the crystal to where dynamic disorder in “frozen out;” in contrast, the multiple static conformations are not temperature-dependent an persist. Analysis of dynamic disorder by itstemperature dependency using X-ray diffraction is an important method for studying protein dynamics.
Crystallization techniques have advanced so much that crystals are obtainable even from less abundant protein. Interesting structures have been reported for (a) proteins in which specific residues have been substituted, (b) antibody-antigen complexes, and (c) viral products such (HIV) that causesacquired immunodeficiency syndrome (AIDS). Approximately virus 30,000 structures have been solved by X-ray diffraction, and the details are stored in the Protein Data Bank, which is readily accessible.
Diffraction of X-rays by a crystal occurs with incident radiation of characteristic wavelength (e.g, cupper, k=1.54A). The X –ray beam is diffracted by the electrons that surround the anatomicnuclei in the crystal, with intensity proportional to the number of electrons around the nucleus. Thus, the technique establishes the electron distribution of the molecule and infers the nuclear distribution. Actual positions of atomic nuclei can be determinate directly by diffraction with neutron beam radiation, and interesting but very expensive technique because it requires a nuclear reactor orparticle accelerator. With the highest resolution now available for x-ray diffraction determination of protein structure, the diffraction from C, N, O and S atoms can be observed. That from hydrogen atoms is not observed due to the low density of electrons-that is, a single electron around a hydrogen nucleus. The diffracted beam is typically detected on photographic film or electronic areadetectors. This permits recording of the amplitude (intensity) of radiation diffracted in a defined orientation. Determination of the phase angles has historically required the placement of heavy atoms (such as iodine, mercury, or lead) in the protein molecule. Modern procedures can often solve the phase problem without use of heavy atoms.
It is convenient to compare x-ray crystallography with light...