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 singlestranded 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 radiationinduced Lys and Phe disordering upon RNA binding directly from the electron density. IntroductionWith 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 now a body of literature on possible strategies to mitigate the effects of RD (e.g. Zeldin, Brockhauser et al., 2013;Bourenkov & Popov, 2010). However, 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 (Shimizu et al., 2007; Liebschner et ISSN 2059-7983 al., 2015) and the efficacy of radical scavengers (Allan et al., 2013) have yet to be resolved.RD manifests in two forms. Global radiation damage is observed within reciprocal space as the overall decay of the summed intensity of reflections detected within the diffraction pattern as dose increases (Garman, 2010;Murray & Garman, 2002). Dose is defined as the a...

Radiation Damage in Macromolecular Crystallography—An Experimentalist’s View

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Phosphorus SAD Phasing for Nucleic Acid Structures: Limitations and Potential

“…For nucleic acid crystallography, radiation damage, while a critically important factor, may not be as serious as it is for protein S-SAD. A recent study of a nucleoprotein complex [109] quantified the per-atom electron density changes over a wide range of radiation dose levels (1.3-25.0 MGy) and showed that RNA is much less sensitive to radiation-induced chemical changes than protein. And, unexpectedly, the normally radiation sensitive Glu and Asp residues within RNA binding pockets appear to have been protected by the association with RNA.…”

Electronic Excitations and Radiation Damage in Macromolecular Crystallography

“…In this work, we will outline how specific damage events in crystallographic models can be interpreted in terms of local structural deformations caused by the population and depopulation of virtual and occupied electronic states in the close vicinity of the Fermi energy of the perfect crystal. Previous reports in the literature have addressed and quantified the local changes, but unfortunately, due to the lack of a unifying model, a quantification of this effect has proved to be very subjective and system dependent (although procedures for radiation damage corrections have been refined empirically to a point where a systematic and automated assessment is possible [1,5,15]). It is our view that single crystal macromolecular crystallography oscillation datasets are unsuitable for assessing the structural changes caused by specific damage, since they rely on information obtained too long (seconds) after the initial interaction of the X-ray beam with the sample.…”