Pressure-induced activation of latent dihydroorotase from Aquifex aeolicus as revealed by high pressure protein crystallography.

Dihydroorotase (DHOase) is involved in the de novo synthesis of pyrimidine in virtually all organisms, and it is usually associated with two other enzymes found in this biosynthetic pathway, carbamylphosphate synthetase and/or aspartate transcarbamylase (ATCase). In the hyperthermophilic bacterium Aquifex aeolicus, ATCase and DHOase are noncovalently associated. Upon dissociation, ATCase keeps its activity entirely while DHOase is totally inactivated.

High-pressure macromolecular crystallography and NMR: status, achievements and prospects.

Biomacromolecules are thermodynamic entities that exist in general as an equilibrium mixture of the basic folded state and various higher-energy substates including all functionally relevant ones. Under physiological conditions, however, the higher-energy substates are usually undetectable on spectroscopy, as their equilibrium populations are extremely low. Hydrostatic pressure gives a general solution to this problem.

Energy resolution of the CdTe-XPAD detector: calibration and potential for Laue diffraction measurements on protein crystals.

The XPAD3S-CdTe, a CdTe photon-counting pixel array detector, has been used to measure the energy and the intensity of the white-beam diffraction from a lysozyme crystal. A method was developed to calibrate the detector in terms of energy, allowing incident photon energy measurement to high resolution (approximately 140 eV), opening up new possibilities in energy-resolved X-ray diffraction. In order to demonstrate this, Laue diffraction experiments were performed on the bending-magnet beamline METROLOGIE at Synchrotron SOLEIL.

A new paradigm for macromolecular crystallography beamlines derived from high-pressure methodology and results.

Biological structures can now be investigated at high resolution by high-pressure X-ray macromolecular crystallography (HPMX). The number of HPMX studies is growing, with applications to polynucleotides, monomeric and multimeric proteins, complex assemblies and even a virus capsid. Investigations of the effects of pressure perturbation have encompassed elastic compression of the native state, study of proteins from extremophiles and trapping of higher-energy conformers that are often of biological interest; measurements of the compressibility of crystals and macromolecules were also performed.

Diffraction structural biology--introductory overview.

An introductory overview to the special issue papers on diffraction structural biology in this issue of the journal.

Flexibility of the Cu,Zn superoxide dismutase structure investigated at 0.57 GPa.

The 2 A resolution crystal structure of bovine erythrocyte Cu,Zn superoxide dismutase (CuZnSOD) has been determined by X-ray diffraction at high pressure (0.57 GPa) and room temperature. At 0.

Structure-function perturbation and dissociation of tetrameric urate oxidase by high hydrostatic pressure.

Structure-function relationships in the tetrameric enzyme urate oxidase were investigated using pressure perturbation. As the active sites are located at the interfaces between monomers, enzyme activity is directly related to the integrity of the tetramer. The effect of hydrostatic pressure on the enzyme was investigated by x-ray crystallography, small-angle x-ray scattering, and fluorescence spectroscopy.

Advances in high-pressure biophysics: status and prospects of macromolecular crystallography.

A survey of the main interests of high pressure for molecular biophysics highlights the possibility of exploring the whole conformational space using pressure perturbation. A better understanding of fundamental mechanisms responsible for the effects of high pressure on biomolecules requires high-resolution molecular information. Thanks to recent instrumental and methodological progress taking advantage of the remarkable adaptation of the crystalline state to hydrostatic compression, pressure-perturbed macromolecular crystallography is now a full-fledged technique applicable to a variety of systems, including large assemblies.

Adaptation of the base-paired double-helix molecular architecture to extreme pressure.

The behaviour of the d(GGTATACC) oligonucleotide has been investigated by X-ray crystallography at 295 K in the range from ambient pressure to 2 GPa (approximately 20,000 atm). Four 3D-structures of the A-DNA form (at ambient pressure, 0.55, 1.

Protein Crystallography at Ultra-Short Wavelengths: Feasibility Study of Anomalous-Dispersion Experiments at the Xenon K-edge.

A protein crystallography experiment at the xenon K-edge (lambda = 0.358 A) has been successfully carried out at the materials science beamline (BL2/ID11) of the ESRF. The samples used in this methodological study were crystals of porcine pancreatic elastase, a 26 kDa protein of known structure.

High-pressure macromolecular crystallography (HPMX): status and prospects.

Recent technical developments, achievements and prospects of high-pressure (HP) macromolecular crystallography (MX) are reviewed. Technical difficulties associated with this technique have been essentially solved by combining synchrotron radiation of ultra-short wavelength, large-aperture diamond anvil cells and new sample-mounting techniques. The quality of diffraction data collected at HP can now meet standards of conventional MX.

High pressure macromolecular crystallography: the 140-MPa crystal structure at 2.3 A resolution of urate oxidase, a 135-kDa tetrameric assembly.

We report the three-dimensional structure determined by high-pressure macromolecular crystallography (HPMX) of a 135-kDa homo-tetrameric enzyme, urate oxidase from Aspergillus flavus complexed with its potent inhibitor 8-azaxanthin. Urate oxidase crystals are quite sensitive to pressure, as three-dimensional order is lost at about 180 MPa. A highly complete 2.

The first crystal structure of a macromolecular assembly under high pressure: CpMV at 330 MPa.

The structure of cubic Cowpea mosaic virus crystals, compressed at 330 MPa in a diamond anvil cell, was refined at 2.8 A from data collected using ultrashort-wavelength (0.331 A) synchrotron radiation.

Metallogenomics and biological X-ray absorption spectroscopy.

An overview of the second special issue of the journal on biological applications of X-ray absorption spectroscopy (BioXAS) is presented. The emphasis is on the study of metalloproteins in the context of structural genomics programmes (metallogenomics).

Using a quasi-parallel X-ray beam of ultrashort wavelength for high-pressure virus crystallography: implications for standard macromolecular crystallography.

Data acquisition from crystals of an icosahedral virus, cowpea mosaic virus (CPMV), was carried out to 2.8 A resolution under an elevated hydrostatic pressure of 330 MPa. This was the first example of a complex macromolecular assembly to be studied by high-pressure crystallography.

A structural genomics initiative on yeast proteins.

A canonical structural genomics programme is being conducted at the Paris-Sud campus area on baker's yeast proteins. Experimental strategies, first results and identified bottlenecks are presented. The actual or potential contributions to the structural genomics of several experimental structure-determination methods are discussed.

Introductory overview: X-ray absorption spectroscopy and structural genomics.

A special Issue of the Journal is presented, dedicated to biological applications of X-ray absorption spectroscopy (BioXAS) and examining the role of this technique in post-genomic biology. The Issue confirms that BioXAS has come of age and it can be expected to make a significant contribution in the structural genomics effort on metalloproteins, which are estimated to make up about 30% of proteins coded by genomes.

Opening the high-pressure domain beyond 2 kbar to protein and virus crystallography--technical advance.

The combined use of a diamond anvil cell and ultrashort-wavelength undulator radiation has allowed the collection of high-resolution diffraction data from protein and virus crystals submitted to hydrostatic pressures beyond 2 kbar. Crystals of cubic cowpea mosaic virus (CPMV) can be compressed to at least 3.5 kbar.

High-pressure protein crystallography (HPPX): instrumentation, methodology and results on lysozyme crystals.

A new set-up and associated methodology for the collection of angle-dispersive diffraction data from protein crystals submitted to high hydrostastic pressure have been developed on beamline ID30 at the ESRF. The instrument makes use of intense X-rays of ultra-short wavelength emitted by two collinear undulators, and combines a membrane-driven diamond-anvil cell mounted on a two-axis goniometer and an imaging-plate scanner. Sharp and clean diffraction pictures from tetragonal crystals of hen egg-white lysozyme (tHEWL) and orthorhombic crystals of bovine erythrocyte Cu, Zn superoxide dismutase (SOD) were recorded at room temperature and pressures up to 0.

Low-resolution phase information in multiple-wavelength anomalous solvent contrast variation experiments.

The basic theory and principles of the multiple-wavelength anomalous solvent-contrast (MASC) method are introduced as a contrast-variation technique for generating low-resolution crystallographic phase information on the envelope of a macromolecule. Experimental techniques and practical considerations concerning the choice of anomalous scatterer, sample preparation and data acquisition are discussed. Test cases of crystals of three proteins of differing molecular weights from 14 kDa through to 173 kDa are illustrated.

Multiwavelength anomalous solvent contrast (MASC): derivation of envelope structure-factor amplitudes and comparison with model values.

A previous article [Fourme et al. (1995). J.

A zipper-like duplex in DNA: the crystal structure of d(GCGAAAGCT) at 2.1 A resolution.

Background: The replication origin of the single-stranded (ss)DNA bacteriophage G4 has been proposed to fold into a hairpin loop containing the sequence GCGAAAGC. This sequence comprises a purine-rich motif (GAAA), which also occurs in conserved repetitive sequences of centromeric DNA. ssDNA analogues of these sequences often show exceptional stability which is associated with hairpin loops or unusual duplexes, and may be important in DNA replication and centromere function.

Exploring hydrophobic sites in proteins with xenon or krypton.

X-ray diffraction is used to study the binding of xenon and krypton to a variety of crystallised proteins: porcine pancreatic elastase; subtilisin Carlsberg from Bacillus licheniformis; cutinase from Fusarium solani; collagenase from Hypoderma lineatum; hen egg lysozyme, the lipoamide dehydrogenase domain from the outer membrane protein P64k from Neisseria meningitidis; urate-oxidase from Aspergillus flavus, mosquitocidal delta-endotoxin CytB from Bacillus thuringiensis and the ligand-binding domain of the human nuclear retinoid-X receptor RXR-alpha. Under gas pressures ranging from 8 to 20 bar, xenon is able to bind to discrete sites in hydrophobic cavities, ligand and substrate binding pockets, and into the pore of channel-like structures. These xenon complexes can be used to map hydrophobic sites in proteins, or as heavy-atom derivatives in the isomorphous replacement method of structure determination.

Molecular structure of the lipoamide dehydrogenase domain of a surface antigen from Neisseria meningitidis.

The protein p64k from the surface of the Neisseria meningitidis bacteria has been characterized as a two-domain protein. It contains a dihydrolipoamide dehydrogenase domain of 482 residues, involving a FAD prosthetic group as a cofactor, and a smaller lipoic acid binding domain of 86 residues. The two domains are joined by a flexible segment rich in alanine and proline residues.