The 1.8 angstrom crystal structure of a statically disordered 17 base-pairRNA duplex: Principles of RNA crystal packing and its effect on nucleic acid structure

The crystal structure of a 17 base RNA oligomer, r(CACCGGAUG GUUCGGUG), has been solved to a resolution of 1.8 Angstrom through a combination of molec ular replacement, multiple isomorphous replacement phasing, and analysis of observed intensity distributions. The oligomer, which forms a stem-loop in solution, crystallized as a pseudo-infinite duplex in spacegroup P321. The asymmetric unit of the crystal contains four superimposed orientations of the duplex that are out of register, such that backbones superimpose, but b ase identity differs. This static disorder was initially discovered by brom inating a single residue per strand in the sequence, and observing four pea ks per strand in difference maps phased with a native molecular replacement solution. The presence of four superimposed duplex "motifs" related by non -crystallographic hypersymmetry was detected by computing [I-2]/[I](2) and Wilson ratios for the observed intensities. The observed ratios matched tho se produced from calculated intensities of a 4-fold statically disordered m odel. Multiconformer simulated annealing refinement against a maximum-likel ihood target incorporating experimental phase information was used to refin e the 4-fold disordered model to an R-free and R of 29.35% and 25.5%, respe ctively. The resulting structure reveals four distinct conformations of the duplex, with an average pairwise backbone rmsd of 2.35 Angstrom. The struc tural differences between the four conformations, which can be attributed t o differences in packing environment, highlight the possible influence of c rystal packing forces on nucleic acid X-ray structures. Analysis of inter-h elical packing between symmetry-related molecules reveals an RNA "zipper" t hat mediates direct phosphate oxygen-2' hydroxyl interactions between close -packed phosphate-sugar backbones. This may be a general mode for RNA terti ary interaction that does not depend on metal ions or primary sequence. (C) 1999 Academic Press.