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

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-damageinduced 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.

SHELIXIR: automation of experimental phasing procedures using SHELXC/D/E

“…minimization of solvent around the crystal mounted on the loop, optimization of absorbed dose and high resolution in the case of UV-RIP. Nevertheless, RIP is a powerful tool because the data can be collected from the same crystal at the same position (de Sanctis et al, 2016). Such an approach can also be used in synchrotron serial crystallography (Foos et al, 2018).…”

X-ray and UV radiation-damage-induced phasing using synchrotron serial crystallography

“…Data were collected at 100 K using a Dectris PILATUS3 2M detector on the ID23-2 microfocus beamline (fixed energy 14.2 keV) at the European Synchrotron Radiation Facility (Flot et al, 2010). UV illumination of insulin crystals was performed using high-power UV-LEDs as described by de Sanctis et al (2016). Data collection was performed using the MeshAndCollect workflow (Zander et al, 2015).…”

“…Both thaumatin (X-ray RIP) and cubic insulin (UV RIP) produced substructures that could be used to produce interpretable phases. Because we have previously shown that down-weighting of the after data-set intensities after an initial scaling can improve all steps of RIP phasing, we evaluated a range of K values (Nanao et al, 2005;de Sanctis et al, 2016;. Because SHELXC/D/E were conceived for pipelines, it is feasible to evaluate a large number of K values automatically via a simple script.…”

“…Specific radiation damage can be induced by X-ray or UV light and affects metals, S atoms in disulfides, thiol linkages and terminal O atoms in carboxylates (with the latter only being induced by X-rays; Burmeister, 2000;Weik et al, 2000;Pattison & Davies, 2006). Specific radiation damage can be of major concern to practitioners of macromolecular crystallography (MX), but in some cases such damage can be used to determine phases experimentally (Ravelli et al, 2003(Ravelli et al, , 2005Zwart et al, 2004;Banumathi et al, 2004;Weiss et al, 2004;Schiltz et al, 2004;Ramagopal et al, 2005;de Sanctis & Nanao, 2012; de Sanctis et al, 2016). This technique is called radiation-damage-induced phasing (RIP) and, by analogy to single isomorphous replacement (SIR), two data sets are used to calculate differences in structure factors (between damaged and less damaged states).…”

“…If the decrease in occupancy at specific sites is large enough and global radiation damage has been minimized, the positions of radiation damage can be determined. UV RIP generally has the advantage of inducing less general global radiation damage compared with X-ray RIP (Nanao & Ravelli, 2006;de Sanctis et al, 2016). When performed on a single crystal or indeed at the same position of single crystals, RIP has the advantage of relatively high isomorphism between the damaged and undamaged data sets.…”