W0171

Twinning versus Non-Crystallographic Symmetry: Problems in Solving the Structure of RbsA AMPPNP Mg²⁺. Matthew C. Clifton†, Huide Zhang‡, Shelley Armstrong†, Mark A. Hermodson‡, Cynthia V. Stauffacher†, †Dept. of Biological Sciences and ‡Dept. of Biochemistry, Purdue Univ., West Lafayette, IN 47907.

Energy-dependent transport of molecules across the membrane is essential for the cell and has been implicated in a number of diseases. To gain an understanding of the mechanism of energy-dependent transport, our laboratory has been investigating the ribose transport complex from E.coli. The complex contains a membrane protein, RbsC, and RbsA, which contains two ATPase domains. The structure of the N-terminal half of RbsA containing ADP and Mg²⁺ was solved to 1.6Å by MIR phasing. To observe the structure of RbsA prior to ATP hydrolysis, data has been collected for RbsA bound to the non-hydrolyzable ATP analog AMPPNP. The new crystals have P222 symmetry, with a=57.28Å, b=91.01Å, and c=111.72Å. Analysis of the systematic absences indicates a screw axis along the a and c axes. Molecular replacement was attempted using the CNS suite of programs, but solutions could never be refined below an R_work of 40% and an R_free of 48%. Attempts at obtaining phase information from a MAD data set have also been unsuccessful using SOLVE. In both cases, it appears as if one molecule in the asymmetric unit is ordered while the other is disordered. Upon inspection of the data, intensity distributions suggest that the crystal is twinned. The unit cell remains the same when processed in a monoclinic setting with β=89.94°. However, there are no twinning laws for orthorhombic or monoclinic crystals with the previously mentioned unit cell parameters. Another possibility is an NCS axis parallel to a crystallographic symmetry axis giving the appearance of a higher symmetry space group. For this to occur, there would have to be a 2₁-screw axis perpendicular to the P2₁ unique axis. Work has begun to try and resolve the processing problems. Structural comparisons between the ADP and AMPPNP bound states will allow investigation of potential changes in the structure of RbsA upon ATP hydrolysis.