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1.  Automatic processing of macromolecular crystallography X-ray diffraction data at the ESRF 
Journal of Applied Crystallography  2013;46(Pt 3):804-810.
A system for the automatic reduction of single- and multi-position macromolecular crystallography data is presented.
The development of automated high-intensity macromolecular crystallography (MX) beamlines at synchrotron facilities has resulted in a remarkable increase in sample throughput. Developments in X-ray detector technology now mean that complete X-ray diffraction datasets can be collected in less than one minute. Such high-speed collection, and the volumes of data that it produces, often make it difficult for even the most experienced users to cope with the deluge. However, the careful reduction of data during experimental sessions is often necessary for the success of a particular project or as an aid in decision making for subsequent experiments. Automated data reduction pipelines provide a fast and reliable alternative to user-initiated processing at the beamline. In order to provide such a pipeline for the MX user community of the European Synchrotron Radiation Facility (ESRF), a system for the rapid automatic processing of MX diffraction data from single and multiple positions on a single or multiple crystals has been developed. Standard integration and data analysis programs have been incorporated into the ESRF data collection, storage and computing environment, with the final results stored and displayed in an intuitive manner in the ISPyB (information system for protein crystallography beamlines) database, from which they are also available for download. In some cases, experimental phase information can be automatically determined from the processed data. Here, the system is described in detail.
PMCID: PMC3654316  PMID: 23682196
automation; data processing; macromolecular crystallography; computer programs
2.  The use of workflows in the design and implementation of complex experiments in macromolecular crystallography 
A powerful and easy-to-use workflow environment has been developed at the ESRF for combining experiment control with online data analysis on synchrotron beamlines. This tool provides the possibility of automating complex experiments without the need for expertise in instrumentation control and programming, but rather by accessing defined beamline services.
The automation of beam delivery, sample handling and data analysis, together with increasing photon flux, diminishing focal spot size and the appearance of fast-readout detectors on synchrotron beamlines, have changed the way that many macromolecular crystallography experiments are planned and executed. Screening for the best diffracting crystal, or even the best diffracting part of a selected crystal, has been enabled by the development of microfocus beams, precise goniometers and fast-readout detectors that all require rapid feedback from the initial processing of images in order to be effective. All of these advances require the coupling of data feedback to the experimental control system and depend on immediate online data-analysis results during the experiment. To facilitate this, a Data Analysis WorkBench (DAWB) for the flexible creation of complex automated protocols has been developed. Here, example workflows designed and implemented using DAWB are presented for enhanced multi-step crystal characterizations, experiments involving crystal re­orientation with kappa goniometers, crystal-burning experiments for empirically determining the radiation sensitivity of a crystal system and the application of mesh scans to find the best location of a crystal to obtain the highest diffraction quality. Beamline users interact with the prepared workflows through a specific brick within the beamline-control GUI MXCuBE.
PMCID: PMC3413211  PMID: 22868763
workflows; automation; data processing; macromolecular crystallography; experimental protocols; characterization; reorientation; radiation damage
3.  Macro-to-Micro Structural Proteomics: Native Source Proteins for High-Throughput Crystallization 
PLoS ONE  2012;7(2):e32498.
Structural biology and structural genomics projects routinely rely on recombinantly expressed proteins, but many proteins and complexes are difficult to obtain by this approach. We investigated native source proteins for high-throughput protein crystallography applications. The Escherichia coli proteome was fractionated, purified, crystallized, and structurally characterized. Macro-scale fermentation and fractionation were used to subdivide the soluble proteome into 408 unique fractions of which 295 fractions yielded crystals in microfluidic crystallization chips. Of the 295 crystals, 152 were selected for optimization, diffraction screening, and data collection. Twenty-three structures were determined, four of which were novel. This study demonstrates the utility of native source proteins for high-throughput crystallography.
PMCID: PMC3290569  PMID: 22393408
4.  Structure of the Dengue Virus Helicase/Nucleoside Triphosphatase Catalytic Domain at a Resolution of 2.4 Å 
Journal of Virology  2005;79(16):10278-10288.
Dengue fever is an important emerging public health concern, with several million viral infections occurring annually, for which no effective therapy currently exists. The NS3 protein from Dengue virus is a multifunctional protein of 69 kDa, endowed with protease, helicase, and nucleoside 5′-triphosphatase (NTPase) activities. Thus, NS3 plays an important role in viral replication and represents a very interesting target for the development of specific antiviral inhibitors. We present the structure of an enzymatically active fragment of the Dengue virus NTPase/helicase catalytic domain to 2.4 Å resolution. The structure is composed of three domains, displays an asymmetric distribution of charges on its surface, and contains a tunnel large enough to accommodate single-stranded RNA. Its C-terminal domain adopts a new fold compared to the NS3 helicase of hepatitis C virus, which has interesting implications for the evolution of the Flaviviridae replication complex. A bound sulfate ion reveals residues involved in the metal-dependent NTPase catalytic mechanism. Comparison with the NS3 hepatitis C virus helicase complexed to single-stranded DNA would place the 3′ single-stranded tail of a nucleic acid duplex in the tunnel that runs across the basic face of the protein. A possible model for the unwinding mechanism is proposed.
PMCID: PMC1182654  PMID: 16051821
5.  Type III secretion proteins PcrV and PcrG from Pseudomonas aeruginosa form a 1:1 complex through high affinity interactions 
BMC Microbiology  2003;3:21.
Pseudomonas aeruginosa, an increasingly prevalent opportunistic pathogen, utilizes a type III secretion system for injection of toxins into host cells in order to initiate infection. A crucial component of this system is PcrV, which is essential for cytotoxicity and is found both within the bacterial cytoplasm and localized extracellularly, suggesting that it may play more than one role in Pseudomonas infectivity. LcrV, the homolog of PcrV in Yersinia, has been proposed to participate in effector secretion regulation by interacting with LcrG, which may act as a secretion blocker. Although PcrV also recognizes PcrG within the bacterial cytoplasm, the roles played by the two proteins in type III secretion in Pseudomonas may be different from the ones suggested for their Yersinia counterparts.
In this work, we demonstrate by native mass spectrometry that PcrV and PcrG expressed and purified from E. coli form a 1:1 complex in vitro. Circular dichroism results indicate that PcrG is highly unstable in the absence of PcrV; in contrast, both PcrV alone and the PcrV:PcrG complex have high structural integrity. Surface plasmon resonance measurements show that PcrV interacts with PcrG with nanomolar affinity (15.6 nM) and rapid kinetics, an observation which is valid both for the full-length form of PcrG (residues 1–98) as well as a form which lacks the C-terminal 24 residues, which are predicted to have low secondary structure content.
PcrV is a crucial component of the type III secretion system of Pseudomonas, but the way in which it participates in toxin secretion is not understood. Here we have characterized the interaction between PcrV and PcrG in vitro, and shown that PcrG is highly unstable. However, it associates readily with PcrV through a region located within its first 74 amino acids to form a high affinity complex. The fact that PcrV associates and dissociates quickly from an unstable molecule points to the transient nature of a PcrV:PcrG complex. These results are in agreement with analyses from pcrV deletion mutants which suggest that PcrV:PcrG may play a different role in effector secretion than the one described for the LcrV:LcrG complex in Yersinia.
PMCID: PMC270082  PMID: 14565848
type III secretion; interactions; pathogen

Results 1-5 (5)