Electron density maps of proteins

Oldfield TJ. Automated tracing of electron-density maps of proteins. Acta Cryst 2003 059 483-91. 

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

The great advantage of neutron diffraction is that small nuclei like hydrogen are readily observed. By comparison with carbon and larger elements, hydrogen is a very weak X-ray diffractor and is typically not observable in electron-density maps of proteins. But hydrogen and its isotope deuterium (2H or D) diffract neutrons very efficiently in comparison with larger elements. 

The Discovery Studio life science platform encompasses capabilities ranging from medicinal chemistry and functional proteomics data to electron density maps of protein-ligand complexes. 

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 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... 

Figure 3.3 Molecular structure of G-protein-coupled receptors. In (a) the electron density map of bovine rhodopsin is shown as obtained by cryoelectron microscopy of two-dimensional arrays of receptors embedded in lipid membrane. The electron densities show seven peaks reflecting the seven a-helices which are predicted to cross the cell membrane. In (b) is shown a helical-wheel diagram of the receptor orientated according to the electron density map shown in (a). The diagram is seen as the receptor would be viewed from outside the cell membrane. The agonist binding pocket is illustrated by the hatched region between TM3, TM5 and TM6. (From Schertler et al. 1993 and Baldwin 1993, reproduced from Schwartz 1996). Reprinted with permission from Textbook of Receptor Pharmacology. Eds Foreman, JC and Johansen, T. Copyright CRC Press, Boca Raton, Florida...

The result is the electron density map of the protein crystal. The final task for the crystallographer is to build the appropriate protein model, i. e., putting amino acid for amino acid into the electron density. Routinely the theoretical amplitudes and phases are calculated from the model and compared to the experimental data in order to check the correctness of model building. The positions of the protein backbone and the amino acid side chains are well defined by X-ray structures at a... 

Filizola, M., Guo, W., Javitch, J. A., and Weinstein, H. (2003) Dimerization in G protein coupled receptors Correlation analysis and electron density maps of rhodopsin from different species suggest subtype-specific interfaces. Biophys. J. 84(2), 1309 Pos Part 2. 

Fig. 16.13. Pore structure of the acetylcholine receptor, based on electron microscopy studies. a) Electron density map of the acetylcholine receptor of the postsynaptic membrane of the electric organ of the ray Torpedo californicus, based on electron microscopy studies. The receptor has a long funnel-like structure in the extracellular region, which narrows at the center of the pore. A smaller funnel structure is observed in the cytoplasmic region of the receptor. Another protein is situated on the cytoplasmic side. The long arrow indicates the direction of ion passage and the small arrow shows the postulated binding site for acetylcholine, b) Schematic representation of the acetylcholine receptor with the M2 hehx as the central block in the ion channel. According to Unwin, (1993).

Nuclease behaves like a typical globular protein in aqueous solution when examined by classic hydrodynamic methods (40) or by measurements of rotational relaxation times for the dimethylaminonaphth-alene sulfonyl derivative (48)- Its intrinsic viscosity, approximately 0.025 dl/g is also consistent with such a conformation. Measurements of its optical rotatory properties, either by estimation of the Moffitt parameter b , or the mean residue rotation at 233 nin, indicate that approximately 15-18% of the polypeptide backbone is in the -helical conformation (47, 48). A similar value is calculated from circular dichroism measurements (48). These estimations agree very closely with the amount of helix actually observed in the electron density map of nuclease, which is discussed in Chapter 7 by Cotton and Hazen, this volume, and Arnone et al. (49). One can state with some assurance, therefore, that the structure of the average molecule of nuclease in neutral, aqueous solution is at least grossly similar to that in the crystalline state. As will be discussed below, this similarity extends to the unique sensitivity to tryptic digestion of a region of the sequence in the presence of ligands (47, 48), which can easily be seen in the solid state as a rather anomalous protrusion from the body of the molecule (19, 49). 

The 2.0 A electron density map of carboxypeptidase A shows three zinc-protein contacts (91). The ligands have been identified as histidine-69, glutamic acid-72 and histidine-196 (91, 101), where the numbers indicate the positions of the residues in the sequence counted from the N-terminal end. The geometry of the complex is irregular but resembles a distorted tetrahedron with an open position directed towards the active site pocket, and presumably occupied by water in the resting enzyme (91). The similarity with the tentative structure of the metal-binding site in carbonic anhydrase is striking. 

Plate 21 Model and portion of electron-density map of bovine Rieske iron-sulfur protein (PDB lrie). The map is contoured around selected residues only. (For discussion, see Chapter 11.) Image SPV/POV-Ray. 

The introduction of mercury(n) into an interchain disulphide bond in a Bence-Jones dimer derived from immunoglobin has been reported.258 An electron density map of the 2-protein using the mercury(n) derivative has been obtained.259 It is possible that this may show where the antigen activity of immunoglobin resides. 

Figure 4.52. Section of the Electron-Density Map of Myoglobin. This section of the electron-density map shows the heme group. The peak of the center of this section corresponds to the position of the iron atom. [From J. C. Kendrew. The three-dimensional structure of a protein molecule. Copyright 1961 by Scientific American, Inc. All rights reserved.]...

R. Huber considered the reaction centre to be a dull photosyntheric protein which cannot do anything. If it would be a receptor, I would be personally interested, he said. D. Oesterhelt generously considered the project as one of the young people. J. Deisenhofer calculated the electron density maps, and we frequendy sat together to try to interpret and to incorporate the new sequence information which we were gathering in D. Oesterhelt s department, into the model. R. Huber suddenly changed his mind when he interpreted the electron density map of phycocyanin (another dull... 

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