JOINT MAXIMUM ENTROPY AND NON-CRYSTALLOGRAPHIC SYMMETRY CONSTRAINTS IN MODEL INDEPENDENT MAP REFINEMENT. Charles W. Carter, Jr., Valentin Ilyin, Xin Huang, and GenPei Li*. Department of Biochemistry and Biophysics, CB 7260 University of North Carolina at Chapel Hill, Chapel Hill, NC 27514 USA and *Department of Physical Chemistry, Beijing University, Beijing, Peoples Republic of China.

We have extended previous studies using entropy maximization under the constraint of solvent flatness (MESF) to refine electron density maps having non-crystallographic symmetry. Bacillus stearothermophilus Tryptophanyl-tRNA synthetase (TrpRS) crystallizes in a family of different polymorphs, either in the absence of biochemically relevant ligands or when bound to such ligands in the absence of high (10mM) ATP concentrations. Ligand-free enzyme grows either as triclinic (P1) or monoclinic (C2) crystals, and liganded complexes grow as monoclinic (P2₁) crystals. Each case presents strong non-crystallographic 32 symmetry in reciprocal space. Placement of the known structure using AMORE has now shown that each asymmetric unit contains either one or two units of three enzyme dimers characterized by 3₁ screw symmetry; all three crystal forms are closely related to space group P3₁21, previously observed at 18 Å resolution in the P2₁ crystals, and thought to be space group P321 (Carter, et al., 1990, Acta Cryst. A46:57-68). This high redundancy provides important phase constraints.

To preserve subtle differences in TrpRS conformation in each crystal, we are pursuing model-independent refinement of each structure, in order to minimize possible model-induced phase biases. From the initial model phases, cycles of phase improvement consist of the following operations: (1) maximum entropy solvent flattening constrained by subsets of roughly the best third of the current phases (Xiang, et al., Acta Cryst D50:193-212); (2) phase permutation, if necessary, involving small numbers of reflections with large renormalized structure factors (Doublié, et al., 1994, Acta Cryst A50:164-182); (3) Non-crystallographic symmetry averaging of centroid q^ME(x) maps obtained by MESF. This process is iterated to convergence. Examples will be described, and compared with examples from studies at low resolution, involving Likelihood ranking of phase sets generated by conventional direct methods and subsequent phase extension. (Supported by NIH GM48519-02 and NSF MCB 9304674)