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Title: RNA protects a nucleoprotein complex against radiation damage

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. We developed a methodology 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 largetrpRNA-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 analysismore » 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. In addition, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.« less
Authors:
 [1] ;  [2] ;  [3] ;  [4] ;  [1] ;  [5] ;  [1]
  1. Univ. of Oxford (United Kingdom). Dept. of Biochemistry
  2. Univ. of Portsmouth (United Kingdom). Inst. of Biomedical and Biomolecular Sciences
  3. Univ. of York (United Kingdom). Dept. of Chemistry
  4. Univ. of Notre Dame, IN (United States). Notre Dame Radiation Lab.
  5. Moscow Inst. of Physics and Technology (MIPT), Moscow (Russian Federation). Lab. of Structural Biology of GPCRs
Publication Date:
Grant/Contract Number:
FC02-04ER15533
Type:
Accepted Manuscript
Journal Name:
Acta Crystallographica. Section D. Structural Biology
Additional Journal Information:
Journal Volume: 72; Journal Issue: 5; Journal ID: ISSN 2059-7983
Publisher:
IUCr
Research Org:
Univ. of Notre Dame, IN (United States)
Sponsoring Org:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; radiation damage; protein–RNA complex; electron difference density; specific damage; decarboxylation
OSTI Identifier:
1345571

Bury, Charles S., McGeehan, John E., Antson, Alfred A., Carmichael, Ian, Gerstel, Markus, Shevtsov, Mikhail B., and Garman, Elspeth F.. RNA protects a nucleoprotein complex against radiation damage. United States: N. p., Web. doi:10.1107/s2059798316003351.
Bury, Charles S., McGeehan, John E., Antson, Alfred A., Carmichael, Ian, Gerstel, Markus, Shevtsov, Mikhail B., & Garman, Elspeth F.. RNA protects a nucleoprotein complex against radiation damage. United States. doi:10.1107/s2059798316003351.
Bury, Charles S., McGeehan, John E., Antson, Alfred A., Carmichael, Ian, Gerstel, Markus, Shevtsov, Mikhail B., and Garman, Elspeth F.. 2016. "RNA protects a nucleoprotein complex against radiation damage". United States. doi:10.1107/s2059798316003351. https://www.osti.gov/servlets/purl/1345571.
@article{osti_1345571,
title = {RNA protects a nucleoprotein complex against radiation damage},
author = {Bury, Charles S. and McGeehan, John E. and Antson, Alfred A. and Carmichael, Ian and Gerstel, Markus and Shevtsov, Mikhail B. and Garman, Elspeth F.},
abstractNote = {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. We developed a methodology 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 largetrpRNA-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. In addition, the method enabled a quantification of the reduction in radiation-induced Lys and Phe disordering upon RNA binding directly from the electron density.},
doi = {10.1107/s2059798316003351},
journal = {Acta Crystallographica. Section D. Structural Biology},
number = 5,
volume = 72,
place = {United States},
year = {2016},
month = {4}
}