Protein crystallography difference maps

Once a suitable crystal is obtained and the X-ray diffraction data are collected, the calculation of the electron density map from the data has to overcome a hurdle inherent to X-ray analysis. The X-rays scattered by the electrons in the protein crystal are defined by their amplitudes and phases, but only the amplitude can be calculated from the intensity of the diffraction spot. Different methods have been developed in order to obtain the phase information. Two approaches, commonly applied in protein crystallography, should be mentioned here. In case the structure of a homologous protein or of a major component in a protein complex is already known, the phases can be obtained by molecular replacement. The other possibility requires further experimentation, since crystals and diffraction data of heavy atom derivatives of the native crystals are also needed. Heavy atoms may be introduced by covalent attachment to cystein residues of the protein prior to crystallization, by soaking of heavy metal salts into the crystal, or by incorporation of heavy atoms in amino acids (e.g., Se-methionine) prior to bacterial synthesis of the recombinant protein. Determination of the phases corresponding to the strongly scattering heavy atoms allows successive determination of all phases. This method is called isomorphous replacement. 

We will now describe some details of the interpretation of the electron-density map that has been calculated. There are two major methods used in protein crystallography to build a model of the molecule from the various features found in an electron-density map. These methods differ from those used for small-molecules because the number of atoms is so large, and because individual atoms are not resolved in most protein crystal structures. A scheme, which is a continuation of Figure 8.10 (Chapter 8), is given in Figure 9.13. 

The problem is a general one of placing a rigid molecular structure in an electron density map in the most objective fashion. There are a number of different types of such problems in protein crystallography. One particular instance is the placement of small substrate (or equally well an inhibitor) structures in poor electron density maps of protein binding sites. Another case is when homologue structures are used in the initial location of new protein structures within the unit cell of poor electron density maps. The location of a known structure within a poor electron density map from a different space group is yet another variant. 

The three-dimensional structure of protein molecules can be experimentally determined by two different methods, x-ray crystallography and NMR. The interaction of x-rays with electrons in molecules arranged in a crystal is used to obtain an electron-density map of the molecule, which can be interpreted in terms of an atomic model. Recent technical advances, such as powerful computers including graphics work stations, electronic area detectors, and... 

Crystal structure of a protein molecule can also be determined by x-ray crystallography. Purified protein is crystallized either by batch methods or vapor diffusion. X-rays are directed at a crystal of protein. The rays are scattered depending on the electron densities in different positions of a protein. Images are translated onto electron density maps and then analyzed computationally to construct a model of the protein. It is especially important for structure-based drug designs. 

At their most elaborate, epitope mapping techniques can provide detailed information on the amino acid residues in a protein antigen, which are in direct contact with the antibody binding site. X-ray crystallography of antibody-antigen complexes can identify contact residues directly and unequivocally, though not surprisingly in view of the effort required, this method is not in routine use. At the other extreme, demonstration by competition enzyme-linked immunosorbent assay (ELISA) methods that two antibodies bind to different sites on... 

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