Macromolecular X-ray crystallography data collection strategies for radiation sensitive proteins

Abstract

Macromolecular X-ray crystallography is the predominant method used for protein structure determination; however, data collection is impeded by sample radiation damage. Global radiation damage manifests as a loss of diffraction intensity, whereas specific radiation damage causes structural changes at radiation sensitive features. This thesis investigates the radiation sensitive, iron binding protein FutA from the cyanobacteria Prochlorococcus MED4. FutA is thought to function primarily as a periplasmic ferric iron (Fe3+) binding protein but may facilitate a second function as an intracellular ferrous iron (Fe2+) binding protein. Structure determination of FutA by X-ray crystallographic methods is impeded by X-ray induced photoreduction of the ferric iron to ferrous iron. Thus, radiation dose-limiting data collection techniques are required to determine an accurate structure of the ferric state. Many radiation dose-limiting data collection techniques have strict sample requirements that specify crystal size, number, and morphology. This thesis discusses the construction of a crystallisation phase diagram for FutA to elucidate the relationship between crystallisation components and the resulting crystal sample. The information provided by the phase diagram allowed suitable FutA samples to be produced for radiation dose-limiting structure determination. As a result, this thesis presents the first radiation-damage-free structure of ferric bound FutA solved using serial femtosecond crystallography (SFX). The sensitivity of FutA to X-ray induced photoreduction was utilised to investigate the potential dual ferric and ferrous iron binding function of FutA. The structural response to photoreduction was determined using dose-series produced by serial synchrotron crystallography (SSX) and was corroborated by the capture of the ferrous state using lowdose, single-crystal X-ray diffraction with a home-source. The photoreduction of FutA was characterised by a repositioning of Arg203 which is thought to stabilise the change in the iron redox state. The ability of FutA to stabilise the change in the redox state from ferric to ferrous iron may be indicative of an in vivo ferrous iron binding function. Many of the radiation-limiting data collection techniques used for FutA structure determination required large amounts of protein material. As a result, these techniques are unsuitable for proteins where the production of crystals is difficult. VMXm is a new micro/nano-focus beamline at Diamond Light Source (UK) which aims to facilitate data collection from crystals limited in size and number. At crystal sizes targeted by VMXm, improvements in crystal lifetime are expected, because global radiation damage is reduced due to the effect of photoelectron escape. In this thesis, the effect of photoelectron escape on the lifetime of microcrystals (3 – 11 µm) was investigated, where crystal lifetime was found to improve by a factor of 1.4 between a 3 µm and 10 µm crystal at 22.3 keV X-ray energy. The measured gains in crystal lifetime presented here corroborate the predicted gains in crystal lifetime by Cowan and Nave (2008)2, when residual liquid surrounding the crystal is accounted for

More information

Identifiers

ORCID for Ivo Tews: orcid.org/0000-0002-4704-1139

Catalogue record

Export record