Radiation-damage-induced phasing: a case study using UV irradiation with light-emitting diodes.

Daniele De Sanctis,Hugo Caserotto,Franck Felisaz,Max H Nanao,Chloe Zubieta

doi:10.1107/s2059798315021658

Abstract

Exposure to X-rays, high-intensity visible light or ultraviolet radiation results in alterations to protein structure such as the breakage of disulfide bonds, the loss of electron density at electron-rich centres and the movement of side chains. These specific changes can be exploited in order to obtain phase information. Here, a case study using insulin to illustrate each step of the radiation-damage-induced phasing (RIP) method is presented. Unlike a traditional X-ray-induced damage step, specific damage is introduced via ultraviolet light-emitting diodes (UV-LEDs). In contrast to UV lasers, UV-LEDs have the advantages of small size, low cost and relative ease of use.

Highlights

Radiation damage during an X-ray diffraction experiment occurs owing to the absorption of X-rays by electron-rich sites in the macromolecule, in certain cases resulting in damage to these specific sites preferentially (Burmeister, 2000; Ravelli & McSweeney, 2000; Leiros et al, 2001)
The R value calculated between the before and after data sets can be useful at this stage, with large values typically indicating an excess of non-isomorphism owing to global radiation damage
Here, we demonstrate the steps necessary for radiation-damageinduced phasing (RIP) and show that ultraviolet light-emitting diodes (UV-LEDs) can be used to introduce sufficient specific radiation damage to determine phases

Summary

Introduction

Radiation damage during an X-ray diffraction experiment occurs owing to the absorption of X-rays by electron-rich sites in the macromolecule, in certain cases resulting in damage to these specific sites preferentially (Burmeister, 2000; Ravelli & McSweeney, 2000; Leiros et al, 2001). By tuning the dose of radiation, specific damage can be induced, creating the opportunity for de novo determination of the ‘substructure’ of radiation-damaged sites (Ravelli et al, 2003) and phasing. This method of phasing was named radiation-damage-induced phasing (RIP) and can be exploited in a manner analogous to the single isomorphous replacement (SIR) method. General radiation damage globally affects the crystal, often resulting in increased Wilson B factors, altered unit-cell parameters, increased mosaicity and decreased resolution (Burmeister, 2000; Ravelli & McSweeney, 2000).

Methods

Results

Conclusion