Protein models refining

Figure 10.3 High resolution data measured with SR for protein model refinement. Electron density at tyr 19 in DPI (a) 1.5A resolution diffractometer data at Cu Ka (b) 1.3 A resolution SRS FAST data. From P. Holden (reported in Glover et al (1988)) and reproduced with permission.

MD production runs are just a continuation of the MD equilibrium and continue up to several nanoseconds. The results from the production runs can be used for docking studies or to determine changes in the secondary or tertiary structure of the target protein. Protein models refined with MD simulations consist of a library of structures with similar backbone geometry, but with different side-chain rotamers. These different structures are especially useful for molecular docking studies when the conformation of the protein and the orientation of the side chains are unknown. 

In molecular pharmacology research an indirect proof of a structural model is possible by functional examinations, e.g., by molecular biological experiments. Well-selected site directed mutagenesis and their functional characterization allows confirmation or rejection of a molecular protein model. The process is organized as an iterative procedure, where the biological answer of suggested mutations is used to refine the model. The iteration continues until the model... 

Lamzin VS, Wilson KS. Automated refinement of protein models. Acta Cryst 1993 049 129-47. 

Perrakis A, Morris R, Lamzin VS. Automated protein model building combined with iterative structure refinement. Nat Struct Biol 1998 6 458-63. 

Refinement takes place by adjusting the model to find closer agreement between the calculated and observed structure factors. For proteins the refinements can yield R-factors in the range of 10-20%. An example taken from reference 10 is instructive. In a refinement of a papain crystal at 1.65-A resolution, 25,000 independent X-ray reflections were measured. Parameters to be refined were the positional parameters (x, y, and z) and one isotropic temperature factor parameter... 

Once the initial phases have heen estimated, a model must he huilt and corrected to give the hest fit of the model to the experimental data. Refinement is an iterative process, cycling between mathematical adjustments to the protein model and examination of the model versus the experimental data and manual rebuilding. 

MR is an ensemble of techniques that aims to place and orientate an approximate molecular model in the unit cell of the crystal being studied. This will provide the starting phases needed to calculate the initial electron density map from which the protein model can be built, either manually by iterative use of reconstruction with molecular graphics packages (Jones et al., 1991) followed by refinement (Murshudov et al., 1997), or automatically if diffraction data up to 2.3 Angstroms or better are available (ARP/wARP (Perrakis et al., 2001), Solve/Resolve (Terwilliger, 2003)). 

Figure 11.3 The ratio of the number of reflections to the number of parameters in the XYZB crystallographic refinement of a protein model as a function of the resolution of the data. The data are assumed to be complete and the solvent content to be 50%.

Lamzin, V. S., Perrakis, A. and Wilson, K. S. (2001). The ARP/wARP suite for automated construction and refinement of protein models. In International Tables for Crystallography. Volume F Crystallography of biological macromolecules, Rossmann, M. G. and Arnold, E. eds., pp. 720-722. Dordrecht, Kluwer Academic Publishers, The Netherlands. 

The amount of published research in the field of self-assembly has exploded in recent years. A computerized search of the literature using self-assembled as the key phrase returned over 14,000 references (another 29,000 references were returned when the concept of self-assembled was searched). There were 2,619 hits in 2002,2,336 hits in 2001, and 1,971 hits in 2000. The first time the phrase self-assembled appeared was in a review of primitive protein models in 1969.15 Refining the search by including molecular electronics with self-assembled produced 356 hits, with the first reviews appearing in 198116 and 1991.17 Since the thoroughness of any literature search is limited by the search terms used, we have used a variety of methods to find relevant literature through March 2003 for the present work. 

Motion in a protein may be modeled by computer using Isaac Newton s equation of motion, / = ma. This modeling requires the three-dimensional coordinates from an X-ray structure analysis as a starting point and some knowledge of interatomic potentials, so that only reasonable interatomic distances will be employed at all stages.Such molecular dynamics calculations lead to a prediction of where atoms will move in a short period of time, and result in the calculation of a time-dependent trajectory of all atoms. Initially each atom is moved in the direction of the force on it from other atoms and then, as each atom moves, its trajectory may change to accommodate this. In addition, this method aids in protein structure refinement,as was described in Chapter 10, although it is important to ensure that the model so refined still fits the electron density map. 

Myelin P2 was the first protein in the iLBP family whose structure was determined (Jones et al., 1988). The crystals contain three copies of the molecule in the asymmetric unit and the structure has now been refined at 2.7 A (Cowan et al., 1993). Because the crystals do not diffract beyond 2.7 A, the precision of the model is not as high as in the other studies and this factor could affect precise hydrogen bonding measurements. In an attempt to overcome this problem, the three molecules were restrained to be similar during the refinement. Only well-determined water molecules were included in the final model. No fatty acid was added to the sample, but later mass spectroscopy measurements indicated that the predominant ligand was oleic acid, which also agreed with the shape of the electron density. This density could not be accounted for by the protein model. 

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