Search tips
Search criteria

Results 1-16 (16)

Clipboard (0)

Select a Filter Below

Year of Publication
Document Types
1.  Radiation damage and derivatization in macromolecular crystallography: a structure factor’s perspective 
Both site-specific radiation damage and heavy-atom derivatization result in small changes to the intensity of reflections. The size of the change owing to each is calculated and compared for individual reflections.
During, or even after, data collection the presence and effects of radiation damage in macromolecular crystallography may not always be immediately obvious. Despite this, radiation damage is almost always present, with site-specific damage occurring on very short time (dose) scales well before global damage becomes apparent. A result of both site-specific radiation damage and derivatization is a change in the relative intensity of reflections. The size and approximate rate of onset of X-ray-induced transformations is compared with the changes expected from derivatization, and strategies for minimizing radiation damage are discussed.
PMCID: PMC4784669  PMID: 26960125
phasing; radiation damage; macromolecular crystallography
2.  A modular and compact portable mini-endstation for high-precision, high-speed fixed target serial crystallography at FEL and synchrotron sources 
Journal of Synchrotron Radiation  2015;22(Pt 6):1372-1378.
A portable sample viewing and alignment system is described which provides fast and reliable motion positioning for fixed target arrays at synchrotrons and free-electron laser sources.
The design and implementation of a compact and portable sample alignment system suitable for use at both synchrotron and free-electron laser (FEL) sources and its performance are described. The system provides the ability to quickly and reliably deliver large numbers of samples using the minimum amount of sample possible, through positioning of fixed target arrays into the X-ray beam. The combination of high-precision stages, high-quality sample viewing, a fast controller and a software layer overcome many of the challenges associated with sample alignment. A straightforward interface that minimizes setup and sample changeover time as well as simplifying communication with the stages during the experiment is also described, together with an intuitive naming convention for defining, tracking and locating sample positions. The setup allows the precise delivery of samples in predefined locations to a specific position in space and time, reliably and simply.
PMCID: PMC4629865  PMID: 26524301
fixed-target serial femtosecond crystallography; portable endstation; motion control; sample delivery
3.  Time-resolved crystallography using the Hadamard Transform 
Nature methods  2014;11(11):1131-1134.
A new method for performing time-resolved X-ray crystallographic experiments based on the Hadamard Transform is proposed and demonstrated. The time-resolution is defined by the underlying periodicity of the probe pulse sequence and the signal to noise is greatly improved compared to the fastest experiments depending on a single pulse. This approach is general and equally applicable to any spectroscopic or imaging measurement where the probe can be encoded.
PMCID: PMC4216935  PMID: 25282611
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.
PMCID: PMC3083913  PMID: 21525644
macromolecular crystallography; single-crystal microspectrophotometry; radiation damage; myoglobin; cytochrome c
5.  Human Cellular Retinaldehyde-Binding Protein Has Secondary Thermal 9-cis-Retinal Isomerase Activity 
Cellular retinaldehyde-binding protein (CRALBP) chaperones 11-cis-retinal to convert opsin receptor molecules into photosensitive retinoid pigments of the eye. We report a thermal secondary isomerase activity of CRALBP when bound to 9-cis-retinal. UV/VIS and 1H-NMR spectroscopy were used to characterize the product as 9,13-dicis-retinal. The X-ray structure of the CRALBP mutant R234W:9-cis-retinal complex at 1.9 Å resolution revealed a niche in the binding-pocket for 9-cis-aldehyde different from that reported for 11-cis-retinal. Combined computational, kinetic, and structural data lead us to propose an isomerization mechanism catalyzed by a network of buried waters. Our findings highlight a specific role of water molecules in both CRALBP-assisted specificity towards 9-cis-retinal and its thermal isomerase activity yielding 9,13-dicis-retinal. Kinetic data from two point mutants of CRALBP support an essential role of Glu202 as the initial proton donor in this isomerization reaction.
PMCID: PMC3936205  PMID: 24328211
6.  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.
PMCID: PMC2651763  PMID: 19240329
single-crystal microspectrophotometry; kinetic crystallography; structural enzymology; radiation damage; macromolecular crystallography; complementary techniques
7.  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.
PMCID: PMC2651761  PMID: 19240326
macromolecular crystallography; flux determination; silicon pin diode; absorbed dose
8.  Exploiting fast detectors to enter a new dimension in room-temperature crystallography 
A departure from a linear or an exponential decay in the diffracting power of macromolecular crystals is observed and accounted for through consideration of a multi-state sequential model.
A departure from a linear or an exponential intensity decay in the diffracting power of protein crystals as a function of absorbed dose is reported. The observation of a lag phase raises the possibility of collecting significantly more data from crystals held at room temperature before an intolerable intensity decay is reached. A simple model accounting for the form of the intensity decay is reintroduced and is applied for the first time to high frame-rate room-temperature data collection.
PMCID: PMC4014120  PMID: 24816094
radiation damage; room temperature; macromolecular crystallography; dose rate
9.  Adhiron: a stable and versatile peptide display scaffold for molecular recognition applications 
We have designed a novel non-antibody scaffold protein, termed Adhiron, based on a phytocystatin consensus sequence. The Adhiron scaffold shows high thermal stability (Tm ca. 101°C), and is expressed well in Escherichia coli. We have determined the X-ray crystal structure of the Adhiron scaffold to 1.75 Å resolution revealing a compact cystatin-like fold. We have constructed a phage-display library in this scaffold by insertion of two variable peptide regions. The library is of high quality and complexity comprising 1.3 × 1010 clones. To demonstrate library efficacy, we screened against the yeast Small Ubiquitin-like Modifier (SUMO). In selected clones, variable region 1 often contained sequences homologous to the known SUMO interactive motif (V/I-X-V/I-V/I). Four Adhirons were further characterised and displayed low nanomolar affinities and high specificity for yeast SUMO with essentially no cross-reactivity to human SUMO protein isoforms. We have identified binders against >100 target molecules to date including as examples, a fibroblast growth factor (FGF1), platelet endothelial cell adhesion molecule (PECAM-1; CD31), the SH2 domain Grb2 and a 12-aa peptide. Adhirons are highly stable and well expressed allowing highly specific binding reagents to be selected for use in molecular recognition applications.
PMCID: PMC4000234  PMID: 24668773
consensus protein; high specificity binding; non-antibody-binding protein; protein–protein interaction; SUMO
10.  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.
PMCID: PMC3852651  PMID: 24311579
radiation damage; protein; disulfide bonds; UV–visible absorption microspectrophotometry; electron paramagnetic resonance
11.  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.
PMCID: PMC3727331  PMID: 23897484
clustering; multiple crystals; BLEND; scaling; merging; multi-crystal data sets
12.  Outrunning free radicals in room-temperature macromolecular crystallography 
A systematic increase in lifetime is observed in room-temperature protein and virus crystals through the use of reduced exposure times and a fast detector.
A significant increase in the lifetime of room-temperature macromolecular crystals is reported through the use of a high-brilliance X-ray beam, reduced exposure times and a fast-readout detector. This is attributed to the ability to collect diffraction data before hydroxyl radicals can propagate through the crystal, fatally disrupting the lattice. Hydroxyl radicals are shown to be trapped in amorphous solutions at 100 K. The trend in crystal lifetime was observed in crystals of a soluble protein (immunoglobulin γ Fc receptor IIIa), a virus (bovine enterovirus serotype 2) and a membrane protein (human A2A adenosine G-protein coupled receptor). The observation of a similar effect in all three systems provides clear evidence for a common optimal strategy for room-temperature data collection and will inform the design of future synchrotron beamlines and detectors for macro­molecular crystallography.
PMCID: PMC4791751  PMID: 22751666
radiation damage; room temperature; dose rate; free radicals
13.  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.
PMCID: PMC3370260  PMID: 22525748
14.  In situ macromolecular crystallography using microbeams 
A sample environment for mounting crystallization trays has been developed on the microfocus beamline I24 at Diamond Light Source. The technical developments and several case studies are described.
Despite significant progress in high-throughput methods in macromolecular crystallography, the production of diffraction-quality crystals remains a major bottleneck. By recording diffraction in situ from crystals in their crystallization plates at room temperature, a number of problems associated with crystal handling and cryoprotection can be side-stepped. Using a dedicated goniometer installed on the microfocus macromolecular crystallography beamline I24 at Diamond Light Source, crystals have been studied in situ with an intense and flexible microfocus beam, allowing weakly diffracting samples to be assessed without a manual crystal-handling step but with good signal to noise, despite the background scatter from the plate. A number of case studies are reported: the structure solution of bovine enterovirus 2, crystallization screening of membrane proteins and complexes, and structure solution from crystallization hits produced via a high-throughput pipeline. These demonstrate the potential for in situ data collection and structure solution with microbeams.
PMCID: PMC4791750  PMID: 22525757
in situ diffraction; microfocus; microbeams; high throughput; room temperature; viruses; membrane proteins; synchrotron radiation
15.  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.
PMCID: PMC3069741  PMID: 21460444
macromolecular crystallography; microcrystallography; X-ray beamlines; synchrotron radiation
16.  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.
PMCID: PMC2935246  PMID: 20823545
microcrystals; mammalian cell expression; autotaxin; ENPP2

Results 1-16 (16)