STRUCTURE AND MOLECULAR-MODEL REFINEMENT OF RHIZOMUCOR-MIEHEI TRIACYLGLYCERIDE LIPASE - A CASE-STUDY OF THE USE OF SIMULATED ANNEALING IN PARTIAL MODEL REFINEMENT

The method of crystallographic refinement with incorporated simulated annealing [Brunger, Kurijan & Karplus (1987). Science, 235, 458-460] was used to refine a partial starting atomic model of Rhizomucor miehei triacylglyceride lipase derived by a combination of multiple isomorphous replacement and molecular replacement methods. Poly-Ala segments (amounting to almost 25% of the molecule) were introduced into the model whenever the original electron density map did not allow confident model building. A single cycle of the simulated annealing protocol resulted in a dramatic improvement of the quality of the electron density map. However, the progress of the refinement was erratic: substantial parts of the poly-Ala segments refined into the corresponding main-chain density, while others were unaffected by the procedure. The structure was refined further using 1.9 angstrom resolution data collected with a Xentronics (Siemens) area detector and employing a combination of simulated annealing and restrained crystallographic least-squares methods. The final crystallographic R factor is 0.129 [reflections within the 7.5-1.9 angstrom shell, for which F > 2-sigma(F)], with good stereochemistry (root-mean-square deviation from ideal bond lengths 0.015 angstrom, and from ideal planes 0.010 angstrom).