Imaging RNA and dynamic protein segments with low-resolution virus crystallography: experimental design, data processing and implications of electron density maps.
Single crystal diffraction data were collected from virus crystals in the resolution range of 270 to 14 A 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 double-stranded DNA bacteriophage HK97 and synthetic Flock House virus-like particles (sFHV). The quality of the low-resolution measurements was confirmed by excellent scaling statistics for both data sets. The sFHV amplitudes between 270 and 90 A resolution were closely similar to independently measured solution scattering data, and to data calculated from the Fourier transform of a uniform density sphere of 315 A diameter. A rotation function computed with the sFHV data between 70 and 20 A resolution was readily interpretable. A uniform density sphere model was used to compute phases for measured amplitudes between 270 and 68 A resolution. The calculated phases were refined and extended to 14 A resolution with real space averaging employing an external mask shape defined by the high-resolution structure. The resulting electron density map displayed regions interpretable as loosely ordered RNA that connected ordered RNA segments seen in a published 3.0 A resolution map. The published high-resolution electron density map lacked data inside 15 A resolution and the interior of the particle in that map appeared hollow. Difference electron density maps corresponding to bulk RNA were computed by subtracting the contribution of the protein shell, based on the available high-resolution atomic model, from either the cryo-electron microscopy density or the low-resolution X-ray density. Features of the RNA were closely similar in the cryo-electron microscopy and X-ray maps, demonstrating the consistency of the two imaging methods. Electron density maps computed at 14 and 6 A resolution with the X-ray amplitudes showed that RNA contributed little to the scattering beyond 14 A resolution.
Tsuruta
H
H
SSRL/SLAC, Stanford University, Stanford, CA, 94309-0210, USA.
Reddy
V S
VS
Wikoff
W R
WR
Johnson
J E
JE
D001435:Bacteriophages / Q000737:chemistry; D002213:Capsid / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000295:instrumentation / Q000379:methods*; D007091:Image Processing, Computer-Assisted; D007304:Insect Viruses / Q000737:chemistry; D008958:Models, Molecular; D012328:RNA Viruses / Q000737:chemistry; D012367:RNA, Viral / Q000737:chemistry*; D012542:Scattering, Radiation; D014764:Viral Proteins / Q000737:chemistry*; D014780:Viruses / Q000737:chemistry*
D012367:RNA, Viral; D014764:Viral Proteins