Proteins interpreting electron density maps

Molecular replacement is where the phases of a known structure are used to determine the structure of a protein that may be identical but crystallized in a different space group or may adopt essentially the same structure (e.g., a homologous protein). Essentially, the calculations find the rotation and translation of the molecule that work with the phases to produce an interpretable electron density map. 

Another useful physical property of the crystal is its density, which can be used to determine several useful microscopic properties, including the protein molecular weight, the proteinlwater ratio in the crystal, and the number of protein molecules in each asymmetric unit (defined later). Molecular weights from crystal density are more accurate than those from electrophoresis or most other methods (except mass spectrometry) and are not affected by dissociation or aggregation of protein molecules. The proteinlwater ratio is used to clarify electron-density maps prior to interpretation (Chapter 7). If the unit cell is symmetric (Chapter 4), it can be subdivided into two or more equivalent parts called asymmetric units (the simplest unit cell contains, or in fact is, one asymmetric unit). For interpreting electron-density maps, it is helpful to know the number of protein molecules per asymmetric unit. 

Figure 12.3 Two-dimensional crystals of the protein bacteriorhodopsin were used to pioneer three-dimensional high-resolution structure determination from electron micrographs. An electron density map to 7 A resolution (a) was obtained and interpreted in terms of seven transmembrane helices (b).

The amplitudes and the phases of the diffraction data from the protein crystals are used to calculate an electron-density map of the repeating unit of the crystal. This map then has to be interpreted as a polypeptide chain with a particular amino acid sequence. The interpretation of the electron-density map is complicated by several limitations of the data. First of all, the map itself contains errors, mainly due to errors in the phase angles. In addition, the quality of the map depends on the resolution of the diffraction data, which in turn depends on how well-ordered the crystals are. This directly influences the image that can be produced. The resolution is measured in A... 

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... 

Greer J. Three dimensional pattern recognition an approach to automated interpretation of electron density maps of proteins. / Mol Biol 1974 82 279-301. 

Leherte, L., Fortier, S., Glasgow, J. and Allen, F.H. (1994) Molecular scene analysis application of a topological approach to the automated interpretation of protein electron-density maps, Acta Cryst., D50, 155-166 and references therein. 

Morris, R. J., Perrakis, A., and Lamzin, V. S. (2003). ARP/wARP and automatic interpretation of protein electron density maps. Method Enzymol. 374, 229-244. 

Eortier, S., Chiverton, A., Glasgow, J. and Leherte, L. (1997). Critical-point analysis in protein electron-density map interpretation. Method Enzymol. Ill, 131-157. 

In Fig. 20, B, R, B2, and R2 are the positions of the base and ribose components of the dinucleotide or independent pyrimidine and purine nucleotides, respectively. The phosphate position pi can be occupied by the 3, 5"-diester (5" refers to the 5 position of R2 in a diester) or the 3 - and 5 -nucleotides, respectively. In the protein crystal a sulfate ion occupied this position in variable degree depending on the pH. Histidine 119 can be in any one of four or more positions depending on various factors. The second base might be in position B2 when it is a pyrimidine. The phosphate of a cyclic substrate or pentacovalent intermediate may be at p,. The position labeled H20 is the position of an isolated peak on the electron density map which is interpreted to be a water molecule, Wi, present in the protein and in the complexes. 

The primary data of protein crystallography yield a three-dimensional electron-density map, which must be interpreted in terms of a three-dimensional model of all atom positions in the protein. Such modeling is usually done by computer graphics. 

Just as the auto mechanic sometimes has parts left over, electron-density maps occasionally show clear, empty density after all known contents of the crystal have been located. Apparent density can appear as an artifact of missing Fourier terms, but this density disappears when a more complete set of data is obtained. Among the possible explanations for density that is not artifactual are ions like phosphate and sulfate from the mother liquor reagents like mercaptoethanol, dithiothreitol, or detergents used in purification or crystallization or cofactors, inhibitors, allosteric effectors, or other small molecules that survived the protein purification. Later discovery of previously unknown but important ligands has sometimes resulted in subsequent interpretation of empty density. 

The electron density distribution for solvent molecules can be improved if the contribution from bulk water to the X-ray scattering is included in the model. This affects the low-angle j X-ray intensity data which are omitted in early stages of the least-squares refinement of protein crystal structures. If they are included in refinement and properly accounted for, the signal-to-noise ratio in the electron density maps is significantly improved and the interpretation of solvent sites is less ambiguous. 

Fig. 10.3 Automatic analysis of ligand electron density. The electron density was interpreted and models of compounds automatically fitted using AutoSolve . Although the binding affinity is weak (AT000037 IC5o=46 pM AT000056 ICso=l mM) the fragments bound into the pocket of Trypsin adopt a clearly ordered conformation. Electron density maps are contoured at 3o- and density due to protein and solvent has been removed for clarity.

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