REFINEMENT OF THE XYLANASE FROM Penicillium simplicissimum AT 1.75 RESOLUTION: assessment of the efficiency of the ARP procedure

Andrea Schmidt1, Victor Lamzin2, Perrakis Anastassis3 and Christoph Kratky1

1 Abteilung für Strukturbiologie, Institut für Physikalische Chemie, Karl-Franzens Universität Graz, Heinrichstraße 28, A-8010 Graz, Austria
2 EMBL Hamburg c/o DESY, Notkestraße 85, 22603 Hamburg, Germany
3 EMBL Grenoble, c/o ILL, Avenue des Martyrs, B.P. 156, 38042 Grenoble Cedex 9, France

The xylanase from the fungus Penicillium simplicissimum is a 32600 kDa protein (302 amino acids) which structurally belongs to the family F / 10 of glycosyl hydrolases. This enzyme from P. Simplicissimum performs exceedingly well in paper bleaching due to a specific mode of action, which could not be explained on the basis of the known molecular and biochemical properties. Therefore we determined the crystal structure. Cryo temperature diffraction data were collected in-house to a resolution of 1.9 and at ELETTRA to a resolution of 1.7 .

Four other family F xylanase structures were available from the Brookhaven Protein Database, and the catalytic domain of the Clostridium thermocellum xylanase was chosen as a starting model for molecular replacement. The procedure (carried out with X-PLOR) yielded one unique solution with an R-factor of 48 % and a correlation coefficient of 33 % after rigid body refinement.

The high resolution data were 100 % complete with an Rmerge of 5.9 for 43712 unique reflections in a resolution range between 10 and 1.75 . This invited the application of the ARP procedure for model rebuilding and refinement, in parallel to the "classic" way of manually fitting the model into the electron density.

The traditional procedure involving alternate graphic rebuilding and refinement steps was quite time consuming: 35 amino acids of the N-terminus were missing from the first map obtained with the MR phases and some loops had to be completely rebuilt. The refinement using X-PLOR and O finally converged to an R-factor of 20.0 % (free R-factor 22.9 %).

The steps taken in the ARP refinement, starting from the crude molecular replacement model, will be outlined and the the various stages of the automated model building and sidechain generation will be illustrated. The final model of this predominantly automatic procedure gave an R-factor of 17.6 %.

The P. simplicissimum xylanase folds into a ( / )8 - barrel (TIM - barrel) with additional loops and helices arranged on the "top". These loop regions are characteristic for a particular xylanase, and they were quite different in the molecular replacement model and the final structure. The structures resulting from both refinement procedures (X-PLOR and ARP) will be compared to the initial model and to each other with respect to differences in loops, in regions of weaker electron density (e.g. evidently mobile sidechains) and in solvent regions.