REFINEMENT OF PROTEIN STRUCTURES

J. Vévodová

National Centre for Biomolecular Research, Facult of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic

The final step of the structure solution is the process of refinement of a macromolecular structure, which can be defined as a gradual adjustment of the real space model parameters so that the maximum possible agreement with the experimental data is achieved.

In all cases of the phase problem solution, the phases obtained are estimated, and often the set of estimates is incomplete. Electron-density maps, calculated using measured amplitudes and first phase estimates, are often difficult or impossible to interpret. As phase improvement and extension proceed, the electron-density maps become clearer and easier to interpret. The iterative process of structure refinement eventually leads to a structure that is in good agreement with the experimental data. The complexity of this task requires a combination of iterative refinement methods based on mathematical principles and of manual building and rebuilding of the three-dimensional real space model of the structure.

The refinement starts either with model derived from an existing structure (MR) or with the most evident part(s) of the structure built according to the electron density maps calculated using the initial phase estimation (MAD, SIR or MIR). In case of model obtained by MR for each (non-hydrogen) atom of the rebuilt model, four parameters are refined: three coordinates (x, y and z) and the isotropic temperature factor frequently called B-value. Also, the new bulk solvent correction parameters and positions of hydrogen atoms are calculated and optimized. After several steps of the refinement, the model is used for calculation of the new phases of the structure factor. These phases together with observed structure factor amplitudes are utilized for calculation of the more accurate electron density maps. The decrease of the R-factors (R_cryst and R_free) after each step of structure refinement indicates the correctness of the structure solution and refinement. The refinement is finished when the structural model explains all significant parts of the electron density maximum and when R-factors values are minimal.

Presently the refinement procedure is involved in the CCP4 software package [4] or in CNS [5] or XPLOR [6] programs. Map fitting could be done by computer graphics. Computer programs such as O [7] or XtalView [8] produce a realistic 3D display of electron density maps.

Keywords used in presentation:

Density modification, rigid body refinement, model rebuilding, least squares and maximum likelihood refinement, energy minimization, simulated annealing, fitting of maps.

References:

1. Rhodes, G. (1993) Crystallography Made Crystal Clear , pp. 29, Academic Press, Saint Diego.

2. Drenth, J. (1994) Principles of Protein X-ray Crystallography, Springer-Verlag, NY.

3. McRee, D.E. (1993) Practical protein Crystallography, Academic Press, Saint Diego.

4. Collaborative Computational Project, Number 4. (1994) Acta Cryst. D50, 760-763.

5. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J. S., Kuszewski, J., Nilges, M. and Pannu, N. S. (1998) Acta Cryst. D54, 905-921.

6. Brünger, A. T. (1992) X-PLOR (version 3.0) Manual, Yale University, New Haven, Connecticut, USA.

7. Jones, T. A., Zou, J. Y., Cowan, S. W. and Kjeldgaard, M. (1991) Acta Cryst. A47, 110-119.

8. McRee, D. E. (1999) J. Structural Biology125, 156-165.