RNA protects a nucleoprotein complex against radiation damage.

Charles S Bury,Ian Carmichael,Elspeth F Garman,Markus Gerstel,John E Mcgeehan,Mikhail B Shevtsov,Alfred A Antson

doi:10.1107/s2059798316003351

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.

Highlights

With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K)
To quantify the exact effects of nucleic acid binding to a protein on Specific radiation damage (SRD) susceptibility, a high-throughput and automated pipeline was created to systematically calculate the electron-density change for every refined atom within the trp RNA-binding attenuation protein (TRAP)–RNA structure as a function of dose
The results provide a further step in the detailed characterization of SRD effects in MX

Summary

Introduction

With the wide use of high-flux third-generation synchrotron sources, radiation damage (RD) has once again become a dominant reason for the failure of structure determination using macromolecular crystallography (MX) in experiments conducted both at room temperature and under cryocooled conditions (100 K). Significant progress has been made in recent years in understanding the inevitable manifestations of X-ray-induced RD within protein crystals, and there is a body of literature on possible strategies to mitigate the effects of RD There is still no general consensus within the field on how to minimize RD during MX data collection, and debates on the dependence of RD progression on incident X-ray energy Dose is defined as the absorbed energy per unit mass of crystal in grays (Gy; 1 Gy = 1 J kgÀ1), and is the metric against which damage progression should be monitored during MX data collection, as opposed to time. At 100 K, an experimental dose limit of 30 MGy has been recommended as an upper limit beyond which the biological information derived from any macromolecular crystal may be compromised (Owen et al, 2006)

Methods

Results

Conclusion