Imaging RNA and dynamic protein segments with low-resolution virus crystallography: Experimental design, data processing and implications of electrondensity maps

Single crystal diffraction data were collected from virus crystals in the r esolution range of 270 to 14 Angstrom using a synchrotron X-ray source and a small-angle scattering instrument adapted for single crystal measurements . Reflections were measured from single crystals of the capsid of the doubl e-stranded DNA bacteriophage HK97 and synthetic Flock House virus-like part icles (sFHV). The quality of the low-resolution measurements was confirmed by excellent scaling statistics for both data sets. The sFHV amplitudes bet ween 270 and 90 Angstrom resolution were closely similar to independently m easured solution scattering data, and to data calculated from the Fourier t ransform of a uniform density sphere of 315 Angstrom diameter. A rotation f unction computed with the sFHV data between 70 and 20 Angstrom resolution w as readily interpretable. A uniform density sphere model was used to comput e phases for measured amplitudes between 270 and 68 Angstrom resolution. Th e calculated phases were refined and extended to 14 Angstrom resolution wit h real space averaging employing an external mask share defined by the high -resolution structure. The resulting electron density map displayed regions interpretable as loosely ordered RNA that connected ordered RNA segments s een in a published 3.0 Angstrom resolution map. The published high-resoluti on electron density map lacked data inside 15 Angstrom resolution and the i nterior of the particle in that map appeared hollow. Difference electron de nsity maps corresponding to bulk RNA were computed by subtracting the contr ibution of the protein shell, based on the available high-resolution atomic model, from either the cryo-electron microscopy density or the low-resolut ion X-ray density. Features of the RNA were closely similar in the cryo-ele ctron microscopy and X-Pay maps, demonstrating the consistency of the two i maging methods. Electron density maps computed at 14 and 6 Angstrom resolut ion with the X-my amplitudes showed that RNA contributed little to the scat tering beyond 14 Angstrom resolution. (C) 1998 Academic Press.