Imaging RNA and dynamic protein segments with low-resolution virus crystallography: experimental design, data...

Area of Science:

• Structural biology
• Virology
• Biophysics

Background:

• The internal structure of virus capsids, particularly the organization of RNA genomes, remains challenging to resolve at high resolution.
• Previous high-resolution studies of Flock House virus-like particles (sFHV) lacked internal density below 15 Å, appearing hollow.
• Understanding genome packaging is crucial for viral assembly and function.

Purpose of the Study:

• To determine the low-resolution structure of RNA within virus capsids using synchrotron X-ray diffraction.
• To compare X-ray diffraction data with cryo-electron microscopy data for consistency.
• To refine and extend electron density maps of virus particles to reveal internal RNA organization.

Main Methods:

• Collected single crystal X-ray diffraction data from sFHV and bacteriophage HK97 capsid crystals.
• Utilized a synchrotron X-ray source and a small-angle scattering instrument adapted for single crystal measurements.
• Employed real-space averaging and difference electron density maps to model and visualize internal RNA structures.

Main Results:

• Obtained interpretable low-resolution data (up to 14 Å) for sFHV and HK97 capsids.
• The electron density map revealed loosely ordered RNA connecting ordered segments, consistent with previous findings.
• Difference maps showed similar RNA features between X-ray and cryo-electron microscopy data, validating both techniques.
• RNA scattering contribution was minimal beyond 14 Å resolution.

Conclusions:

• Low-resolution X-ray diffraction is effective for visualizing internal viral RNA structures.
• The study confirms the consistency of structural information obtained from X-ray diffraction and cryo-electron microscopy.
• The findings provide insights into genome packaging within virus capsids.