title: RNA protects a nucleoprotein complex against radiation damage.
abstract: Radiation damage during macromolecular X-ray crystallographic data collection is still the main impediment for many macromolecular structure determinations. Even when an eventual model results from the crystallographic pipeline, the manifestations of radiation-induced structural and conformation changes, the so-called specific damage, within crystalline macromolecules can lead to false interpretations of biological mechanisms. Although this has been well characterized within protein crystals, far less is known about specific damage effects within the larger class of nucleoprotein complexes. Here, a methodology has been developed whereby per-atom density changes could be quantified with increasing dose over a wide (1.3-25.0 MGy) range and at higher resolution (1.98 Å) than the previous systematic specific damage study on a protein-DNA complex. Specific damage manifestations were determined within the large trp RNA-binding attenuation protein (TRAP) bound to a single-stranded RNA that forms a belt around the protein. Over a large dose range, the RNA was found to be far less susceptible to radiation-induced chemical changes than the protein. The availability of two TRAP molecules in the asymmetric unit, of which only one contained bound RNA, allowed a controlled investigation into the exact role of RNA binding in protein specific damage susceptibility. The 11-fold symmetry within each TRAP ring permitted statistically significant analysis of the Glu and Asp damage patterns, with RNA binding unexpectedly being observed to protect these otherwise highly sensitive residues within the 11 RNA-binding pockets distributed around the outside of the protein molecule. Additionally, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.
authors.affiliation: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England.
authors.forename: Charles S
authors.initials: CS
authors.lastname: Bury
authors.affiliation: Molecular Biophysics, Institute of Biomedical and Biomolecular Sciences, University of Portsmouth, King Henry I Street, Portsmouth PO1 2DY, England.
authors.forename: John E
authors.initials: JE
authors.lastname: McGeehan
authors.affiliation: York Structural Biology Laboratory, Department of Chemistry, University of York, York Y010 5DD, England.
authors.forename: Alfred A
authors.initials: AA
authors.lastname: Antson
authors.affiliation: Notre Dame Radiation Laboratory, University of Notre Dame, Notre Dame, IN 46556, USA.
authors.forename: Ian
authors.initials: I
authors.lastname: Carmichael
authors.affiliation: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England.
authors.forename: Markus
authors.initials: M
authors.lastname: Gerstel
authors.affiliation: Laboratory of Structural Biology of GPCRs, Moscow Institute of Physics and Technology, Dolgoprudniy 141700, Russian Federation.
authors.forename: Mikhail B
authors.initials: MB
authors.lastname: Shevtsov
authors.affiliation: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, England.
authors.forename: Elspeth F
authors.initials: EF
authors.lastname: Garman
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism; D018360:Crystallography, X-Ray / Q000379:methods*; D001411:Geobacillus stearothermophilus / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation / Q000528:radiation effects*; D012313:RNA / Q000737:chemistry* / Q000378:metabolism; D016601:RNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014965:X-Rays / Q000009:adverse effects
chemical_list: D001426:Bacterial Proteins; C079092:MtrB protein, Bacteria; D016601:RNA-Binding Proteins; D014157:Transcription Factors; D012313:RNA
keywords: decarboxylation; electron difference density; protein–RNA complex; radiation damage; specific damage