HOW TO AVOID OVERFITTING IN REFINEMENT OF STRUCTURES WITH APPROXIMATE NON-CRYSTALLOGRAPHIC SYMMETRY. HIGH RESOLUTION STRUCTURE REFINEMENT OF GLUTAMINE SYNTHETASE REVEALS EVIDENCE FOR HOMOTROPIC COOPERATIVE BINDING. Gaston M.U. Pfluegl, Harindarpal Gill & David Eisenberg. Molecular Biology Institute, UCLA, Los Angeles, CA 90095-1570, USA.

Glutamine Synthetase (GS) is a key enzyme in nitrogen metabolism. GS from S.typhimurium is a stable complex of large size, consisting of 12 identical chains assembled into a dodecamer with 62 symmetry (Mr ~ 620 KDa; 12 times 468 amino acid residues). In the presence of ADP, GS crystallizes in space group C2 with 1 dodecamer per asymmetric unit. From the association constant of ADP for GS, about two ADP should be bound per dodecamer under crystallization conditions.

The previous 2.8 Å model for GS has been refined with strict non-crystallographic symmetry restraints (12-fold averaging). In this model, three regions were invisible and some of the active site residues showed disorder. No density was found for ADP in this model.

The present work is based on a 2.5 Å cryo synchrotron data set from a single crystal (98% complete; 200,000 reflections; 10 times redundancy). The new model was refined with 12 independent but constrained subunits and shows density for two bound ADP molecules in two adjacent active sites. This density was not visible in the 12- fold averaged structure. Due to selective binding of ADP within the dodecamer, the GS molecule deviates from exact 62 symmetry. This explains the preference of the C2 crystal form over hexagonal crystal forms which are observed for higher and lower ADP concentrations.

The use of R-free to monitor data overfitting when releasing strict non-crystallographic symmetry will be discussed and data for the choice of the reflections for the TEST-set (not well established for structures with non-crystallographic symmetry) will be presented.

Binding of two ADP molecules in an up-down fashion in the dodecamer sandwich, as seen in the new model, supports the biochemical finding of homotropic cooperative binding of substrate, for which kinetic data will be presented along with the crystallographic data.