Radiation damage within nucleoprotein complexes studied by macromolecular X-ray crystallography

Charles S Bury,Ian Carmichael,John E Mcgeehan,Elspeth F Garman

doi:10.1016/j.radphyschem.2016.05.023

Abstract

In X-ray crystallography, for the determination of the 3-D structure of macromolecules, radiation damage is still an inherent problem at modern third generation synchrotron sources, even when utilising cryo-crystallographic techniques (sample held at 100K). At doses of just several MGy, at which a typical diffraction dataset is collected, site-specific radiation-induced chemical changes are known to manifest within protein crystals, and a wide body of literature is now devoted to understanding the mechanisms behind such damage. Far less is known regarding radiation-induced damage to crystalline nucleic acids and the wider class of nucleoprotein complexes during macromolecular X-ray crystallography (MX) data collection. As the MX structural biology community now strives to solve structures for increasingly larger and complex macromolecular assemblies, it essential to understand how such structures are affected by the X-ray radiation used to solve them. The purpose of this review is to summarise advances in the field of specific damage to nucleoprotein complexes and to present case studies of MX damage investigations on both protein-DNA (C.Esp1396I) and protein-RNA (TRAP) complexes. To motivate further investigations into MX damage mechanisms within nucleoprotein complexes, current and emerging protocols for investigating specific damage within Fobs(n)−Fobs(1) electron density difference maps are discussed.

Highlights

In X-ray crystallography, for the determination of the 3-D structure of macromolecules, radiation damage is still an inherent problem at modern third generation synchrotron sources, even when utilising cryocrystallographic techniques
We suggest that the relative protection provided by RNA-binding to Glu-36 and Asp-39 directly correlates with the functional importance of each residue in high affinity RNA-binding to Trp RNA-binding attenuation protein (TRAP)
It could be postulated that in the crystal environment at 100 K, tight crystal packing could obstruct the development of large nucleic acid deformation events reproducibly throughout crystal unit cells

Summary

Introduction

Of the molecular interactions underpinning the function of highly biologically-important macromolecules (Ferreira et al, 2004; Voorhees et al, 2009). For the C.Esp1396I complex, within the first difference map (Fobs(2) À Fobs(1), 6.2 MGy) clear density loss was localised around acidic residue side-chain carboxyl groups, indicative of radiation-induced decarboxylation, and around methylthio sidechain groups, consistent with Met CH3–S covalent bond cleavage (Fig. 2(a)-(b)) Such observations were highly consistent with previous reports of protein SRD (Weik et al, 2000) (no disulphide bonds are present within this protein). Positive electron density accumulation was observed with increasing dose near the T24 and A25 bases; this is consistent with mechanisms of LEE attachment to nucleobases as suggested within oligonucleotide film studies (Alizadeh et al, 2013), or base modification induced by close proximity solvent free radicals (Cadet et al, 1999) (for example, hydroxyl radical binding to carbon 6 in T24) The location of such a SSB correlates with a DNA region that is both AT-rich and under significant strain as a consequence of large-scale deformation due to protein binding. Esp1396I, the relevance of such protective mechanisms is unclear, and whether protein is intrinsically more susceptible to X-ray induced damage, or whether the protein scavenges electrons to protect DNA remains to be determined

The TRAP-RNA complex: a model protein-RNA MX damage investigation

Findings

Conclusion