Structure determination of Naudaurelia capensis omega virus

The structure of Nudaurelia capensis omega virus (N omega V), a single-stra nded RNA virus, was determined to 2.8 Angstrom resolution. Triclinic crysta ls (a = 413.6, b = 410.2, c = 419.7 Angstrom, alpha = 59.13, beta = 58.9, g amma = 64.0 degrees) diffracted X-rays beyond 2.7 A resolution. The unit ce ll contained one icosahedral virus particle, providing 60-fold noncrystallo graphic symmetry (n.c.s.) and structural redundancy. The particle orientati on in the unit cell was determined by self-rotation function analyses. Init ial phases to 18 Angstrom resolution were derived from a hollow spherical m odel of 192 Angstrom outer radius and 139 Angstrom inner radius, filled wit h uniform electron density. Radii of the model were determined by maximizin g the correlation of the model-based calculated data with the low-resolutio n X-ray diffraction and solution-scattering data. Phases were refined by 60 -fold noncrystallographic electron-density averaging and extended in small steps to a resolution of 5 Angstrom. Time phases obtained represented a mix ture of four different phase sets, each consistent with the icosahedral sym metry constraints. The resulting electron density was not interpretable. A difference Fourier map computed with the native and an isomorphous heavy-at om derivative data sets and phases refined by real-space averaging was inte rpretable only if data within the 10 Angstrom resolution shell were used. M aps calculated with data significantly higher than 10 Angstrom resolution f ailed to display a constellation of heavy-atom sites consistent with the T= 4 icosahedral symmetry. Attempts to extend the phases beyond 10 Angstrom r esolution, starting with either phases based on a model or single isomorpho us replacement, were unsuccessful. Successful phase extension was achieved by computing the phases for the higher resolution reflections from a partia l atomic Model (poly gly) built into the averaged 10 Angstrom electron-dens ity map. Phases from this model served as the starting point for n.c.s. pha se refinement and extension to slightly higher resolution. The atomic model was improved at each extension interval and these phases were used for the subsequent phase calculation and extension. The entire polypeptide backbon e corresponding to the N omega V structure was built into the map at 4 Angs trom. The same procedure for phase refinement was used to extend the phases to 2.8 Angstrom in small increments of resolution. The overall molecular a veraging R factor and correlation coefficient at 2.8 Angstrom resolution we re 18.4% and 0.87, respectively.