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1.  Insights into the mechanism of X-ray-induced disulfide-bond cleavage in lysozyme crystals based on EPR, optical absorption and X-ray diffraction studies 
Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage, to confirm a multi-track radiation-damage process and to develop a model of that process.
Electron paramagnetic resonance (EPR) and online UV–visible absorption microspectrophotometry with X-ray crystallography have been used in a complementary manner to follow X-ray-induced disulfide-bond cleavage. Online UV–visible spectroscopy showed that upon X-irradiation, disulfide radicalization appeared to saturate at an absorbed dose of approximately 0.5–0.8 MGy, in contrast to the saturating dose of ∼0.2 MGy observed using EPR at much lower dose rates. The observations suggest that a multi-track model involving product formation owing to the interaction of two separate tracks is a valid model for radiation damage in protein crystals. The saturation levels are remarkably consistent given the widely different experimental parameters and the range of total absorbed doses studied. The results indicate that even at the lowest doses used for structural investigations disulfide bonds are already radicalized. Multi-track considerations offer the first step in a comprehensive model of radiation damage that could potentially lead to a combined computational and experimental approach to identifying when damage is likely to be present, to quantitate it and to provide the ability to recover the native unperturbed structure.
doi:10.1107/S0907444913022117
PMCID: PMC3852651  PMID: 24311579
radiation damage; protein; disulfide bonds; UV–visible absorption microspectrophotometry; electron paramagnetic resonance
2.  Clustering procedures for the optimal selection of data sets from multiple crystals in macromolecular crystallography 
A systematic approach to the scaling and merging of data from multiple crystals in macromolecular crystallography is introduced and explained.
The availability of intense microbeam macromolecular crystallography beamlines at third-generation synchrotron sources has enabled data collection and structure solution from microcrystals of <10 µm in size. The increased likelihood of severe radiation damage where microcrystals or particularly sensitive crystals are used forces crystallographers to acquire large numbers of data sets from many crystals of the same protein structure. The associated analysis and merging of multi-crystal data is currently a manual and time-consuming step. Here, a computer program, BLEND, that has been written to assist with and automate many of the steps in this process is described. It is demonstrated how BLEND has successfully been used in the solution of a novel membrane protein.
doi:10.1107/S0907444913012274
PMCID: PMC3727331  PMID: 23897484
clustering; multiple crystals; BLEND; scaling; merging; multi-crystal data sets
3.  X-ray-excited optical luminescence of protein crystals: a new tool for studying radiation damage during diffraction data collection 
During X-ray irradiation protein crystals radiate energy in the form of small amounts of visible light. This is known as X-ray-excited optical luminescence (XEOL). The XEOL of several proteins and their constituent amino acids has been characterized using the microspectrophotometers at the Swiss Light Source and Diamond Light Source. XEOL arises primarily from aromatic amino acids, but the effects of local environment and quenching within a crystal mean that the XEOL spectrum of a crystal is not the simple sum of the spectra of its constituent parts. Upon repeated exposure to X-rays XEOL spectra decay non-uniformly, suggesting that XEOL is sensitive to site-specific radiation damage. However, rates of XEOL decay were found not to correlate to decays in diffracting power, making XEOL of limited use as a metric for radiation damage to protein crystals.
doi:10.1107/S0907444912002946
PMCID: PMC3370260  PMID: 22525748
4.  Revealing low-dose radiation damage using single-crystal spectroscopy 
Journal of Synchrotron Radiation  2011;18(Pt 3):367-373.
Data on the rapid reduction of haem proteins in the X-ray beam at synchrotron sources are presented. The use of single-crystal spectroscopy to detect these changes and their implication for diffraction data collection from oxidized species is also discussed.
The structural information and functional insight obtained from X-ray crystallography can be enhanced by the use of complementary spectroscopies. Here the information that can be obtained from spectroscopic methods commonly used in conjunction with X-ray crystallography and best-practice single-crystal UV-Vis absorption data collection are briefly reviewed. Using data collected with the in situ system at the Swiss Light Source, the time and dose scales of low-dose X-ray-induced radiation damage and solvated electron generation in metalloproteins at 100 K are investigated. The effect of dose rate on these scales is also discussed.
doi:10.1107/S0909049511004250
PMCID: PMC3083913  PMID: 21525644
macromolecular crystallography; single-crystal microspectrophotometry; radiation damage; myoglobin; cytochrome c
5.  The design of macromolecular crystallography diffraction experiments 
Thoughts about the decisions made in designing macromolecular X-ray crystallography experiments at synchrotron beamlines are presented.
The measurement of X-ray diffraction data from macro­molecular crystals for the purpose of structure determination is the convergence of two processes: the preparation of diffraction-quality crystal samples on the one hand and the construction and optimization of an X-ray beamline and end station on the other. Like sample preparation, a macromolecular crystallography beamline is geared to obtaining the best possible diffraction measurements from crystals provided by the synchrotron user. This paper describes the thoughts behind an experiment that fully exploits both the sample and the beamline and how these map into everyday decisions that users can and should make when visiting a beamline with their most precious crystals.
doi:10.1107/S0907444911007608
PMCID: PMC3069741  PMID: 21460444
macromolecular crystallography; microcrystallography; X-ray beamlines; synchrotron radiation
6.  Mammalian cell expression, purification, crystallization and microcrystal data collection of autotaxin/ENPP2, a secreted mammalian glycoprotein 
Autotaxin, a four-domain ∼100 kDa mammalian glycoprotein, was expressed in stably transfected mammalian cells, purified from the medium and crystallized. Diffraction data from micrometre-thick crystal plates were collected on various European synchrotron beamlines and are presented and analysed.
Autotaxin (ATX or ENPP2) is a secreted glycosylated mammalian enzyme that exhibits lysophospholipase D activity, hydrolyzing lysophosphatidylcholine to the signalling lipid lysophosphatidic acid. ATX is an ∼100 kDa multi-domain protein encompassing two N-terminal somatomedin B-like domains, a central catalytic phosphodiesterase domain and a C-terminal nuclease-like domain. Protocols for the efficient expression of ATX from stably transfected mammalian HEK293 cells in amounts sufficient for crystallographic studies are reported. Purification resulted in protein that crystallized readily, but various attempts to grow crystals suitable in size for routine crystallographic structure determination were not successful. However, the available micrometre-thick plates diffracted X-rays beyond 2.0 Å resolution and allowed the collection of complete diffraction data to about 2.6 Å resolution. The problems encountered and the current advantages and limitations of diffraction data collection from thin crystal plates are discussed.
doi:10.1107/S1744309110032938
PMCID: PMC2935246  PMID: 20823545
microcrystals; mammalian cell expression; autotaxin; ENPP2
7.  A new on-axis multimode spectrometer for the macromolecular crystallography beamlines of the Swiss Light Source 
Journal of Synchrotron Radiation  2009;16(Pt 2):173-182.
Complementary techniques greatly aid the interpretation of macromolecule structures to yield functional information, and can also help to track radiation-induced changes. A new on-axis spectrometer being integrated into the macromolecular crystallography beamlines of the Swiss Light Source is presented.
X-ray crystallography at third-generation synchrotron sources permits tremendous insight into the three-dimensional structure of macromolecules. Additional information is, however, often required to aid the transition from structure to function. In situ spectroscopic methods such as UV–Vis absorption and (resonance) Raman can provide this, and can also provide a means of detecting X-ray-induced changes. Here, preliminary results are introduced from an on-axis UV–Vis absorption and Raman multimode spectrometer currently being integrated into the beamline environment at X10SA of the Swiss Light Source. The continuing development of the spectrometer is also outlined.
doi:10.1107/S0909049508040120
PMCID: PMC2651763  PMID: 19240329
single-crystal microspectrophotometry; kinetic crystallography; structural enzymology; radiation damage; macromolecular crystallography; complementary techniques
8.  Determination of X-ray flux using silicon pin diodes 
Journal of Synchrotron Radiation  2009;16(Pt 2):143-151.
Accurate measurement of photon flux from an X-ray source is a parameter required to calculate the dose absorbed by a sample. The development of a model for determining the photon flux incident on pin diodes, and experiments to test this model, are described for incident energies between 4 and 18 keV used in macromolecular crystallography.
Accurate measurement of photon flux from an X-ray source, a parameter required to calculate the dose absorbed by the sample, is not yet routinely available at macromolecular crystallography beamlines. The development of a model for determining the photon flux incident on pin diodes is described here, and has been tested on the macromolecular crystallography beamlines at both the Swiss Light Source, Villigen, Switzerland, and the Advanced Light Source, Berkeley, USA, at energies between 4 and 18 keV. These experiments have shown that a simple model based on energy deposition in silicon is sufficient for determining the flux incident on high-quality silicon pin diodes. The derivation and validation of this model is presented, and a web-based tool for the use of the macromolecular crystallography and wider synchrotron community is introduced.
doi:10.1107/S0909049508040429
PMCID: PMC2651761  PMID: 19240326
macromolecular crystallography; flux determination; silicon pin diode; absorbed dose

Results 1-8 (8)