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Biomedical subjects

G Bricogne

Publications and source records attributed to G Bricogne.

At least 19 recordsLinked to original sources
PubMed2006

Modelling and refining site-specific radiation damage in SAD/MAD phasing.

Site-specific radiation damage on anomalously scattering sites can be used to generate additional phase information in standard single- or multi-wavelength anomalous diffraction (SAD or MAD) experiments. In this approach the data are kept unmerged, down to the Harker construction, and the evolution of site-specific radiation damage as a function of X-ray irradiation is explicitly modelled and refined in real space. Phasing power is generated through the intensity differences of symmetry-related reflections or repeated measurements of the same reflection recorded at different X-ray doses. In the present communication the fundamentals of this approach are reviewed and different models for the description of site-specific radiation damage are presented. It is shown that, in more difficult situations, overall radiation damage may unfold on a time scale that is similar to the evolution of site-specific radiation damage or to the total time that is required to record a complete data set. In such cases the quality of the phases will ultimately be limited by the effects of overall radiation damage.

Computer Simulation↗
PubMed2004

Refinement of severely incomplete structures with maximum likelihood in BUSTER-TNT.

BUSTER-TNT is a maximum-likelihood macromolecular refinement package. BUSTER assembles the structural model, scales observed and calculated structure-factor amplitudes and computes the model likelihood, whilst TNT handles the stereochemistry and NCS restraints/constraints and shifts the atomic coordinates, B factors and occupancies. In real space, in addition to the traditional atomic and bulk-solvent models, BUSTER models the parts of the structure for which an atomic model is not yet available ('missing structure') as low-resolution probability distributions for the random positions of the missing atoms. In reciprocal space, the BUSTER structure-factor distribution in the complex plane is a two-dimensional Gaussian centred around the structure factor calculated from the atomic, bulk-solvent and missing-structure models. The errors associated with these three structural components are added to compute the overall spread of the Gaussian. When the atomic model is very incomplete, modelling of the missing structure and the consistency of the BUSTER statistical model help structure building and completion because (i) the accuracy of the overall scale factors is increased, (ii) the bias affecting atomic model refinement is reduced by accounting for some of the scattering from the missing structure, (iii) the addition of a spatial definition to the source of incompleteness improves on traditional Luzzati and sigmaA-based error models and (iv) the program can perform selective density modification in the regions of unbuilt structure alone.

Algorithms↗
PubMed2004

Phasing in the presence of severe site-specific radiation damage through dose-dependent modelling of heavy atoms.

The case of a brominated RNA crystal structure determination in which standard three-wavelength MAD phasing was unsuccessful because of fast X-ray-induced debromination was reinvestigated [Ennifar et al. (2002), Acta Cryst. D58, 1262-1268]. It was found that if the data are kept unmerged and if a dose-stamp is associated with each reflection measurement, dose-dependent occupancies can be refined for the Br atoms. Such a parametrization has been implemented in the macromolecular phasing program SHARP. Refining such dose-dependent occupancies on an unmerged data set gave a dramatic improvement, even for SAD phases from only the first wavelength (peak), and resulted in a good electron-density map after solvent flattening. The adverse effect of radiation damage has been turned into a beneficial one. The crucial difference is made by the use of unmerged data: phasing power is generated through the intensity differences of symmetry-related reflections recorded at different doses, i.e. corresponding to different states of the X-ray-induced debromination. This approach should prove useful in all situations of experimental phasing where site-specific radiation damage occurs unavoidably and undesirably and not only in cases in which radiation damage is purposely being created in order to demonstrate its potential usefulness.

Binding Sites↗
PubMed2003

Generation, representation and flow of phase information in structure determination: recent developments in and around SHARP 2.0.

The methods for treating experimental data in the isomorphous replacement and anomalous scattering methods of macromolecular phase determination have undergone considerable evolution since their inception 50 years ago. The successive formulations used are reviewed, from the most simplistic viewpoint to the most advanced, including the exploration of some blind alleys. A new treatment is proposed and demonstrated for the improved encoding and subsequent exploitation of phase information in the complex plane. It is concluded that there is still considerable scope for further improvements in the statistical analysis of phase information, which touch upon numerous fundamental issues related to data processing and experimental design.

Bayes Theorem↗
PubMed2003

'MAD'ly phasing the extracellular domain of the LDL receptor: a medium-sized protein, large tungsten clusters and multiple non-isomorphous crystals.

The crystal structure of the extracellular domain of the LDL receptor (LDL-R) was determined in a MAD experiment using 12-tungstophosphate clusters as anomalous scatterers. While useful for phasing, the tungsten clusters rendered the crystals radiation-sensitive and non-isomorphous and profoundly altered the diffraction data, causing complications. The work is presented as a case study for phasing a medium-sized protein (700 residues) at low resolution (4 A) with multiple non-isomorphous crystals containing 31 W atoms in the asymmetric unit.

Animals↗
PubMed2002

A novel combination of two classic catalytic schemes.

The crystal structure of an alkaline Bacillus cellulase catalytic core, from glucoside hydrolase family 5, reveals a novel combination of the catalytic machinery of two classic textbook enzymes. The enzyme has the expected two glutamate residues in close proximity to one another in the active-site that are typical of retaining cellulases. However, the proton donor, glutamate 139 is also unexpectedly a member of a serine-histidine-glutamate catalytic triad, forming a novel combination of catalytic machineries. Structure and sequence analysis of glucoside hydrolase family 5 reveal that the triad is highly conserved, but with variations at the equivalent of the serine position. We speculate that the purpose of this novel catalytic triad is to control the protonation of the acid/base glutamate, facilitating the first step of the catalytic reaction, protonation of the substrate, by the proton donor glutamate. If correct, this will be a novel use for a catalytic triad.

Bacillus↗
PubMed2001

High-resolution experimental phases for tryptophanyl-tRNA synthetase (TrpRS) complexed with tryptophanyl-5'AMP.

Native data, anomalous data at three wavelengths and an independent peak-wavelength data set for SeMet-substituted protein have been collected from cryoprotected crystals of the TrpRS-adenylate product (TAM) complex to a resolution limit of 1.7 A. Independent phase sets were developed using SHARP and improved by solvent flipping with SOLOMON using molecular envelopes derived from experimental densities for, respectively, peak-wavelength SAD data from four different crystals, MAD data and their M(S)IRAS combinations with native data. Hendrickson-Lattman phase-probability coefficients from each phase set were used in BUSTER to drive maximum-likelihood refinements of well defined parts of the previously refined room-temperature 2.9 A structure. Maximum-entropy completion followed by manual rebuilding was then used to generate a model for the missing segments, bound ligand and solvent molecules. Surprisingly, peak-wavelength SAD experiments produced the smallest phase errors relative to the refined structures. Selenomethionylated models deviate from one another by 0.25 A and from the native model by 0.38 A, but all have r.m.s. deviations of approximately 1.0 A from the 2.9 A model. Difference Fourier calculations between amplitudes from the 300 K experiment and the new amplitudes at 100 K using 1.7 A model phases show no significant structural changes arising from temperature variation or addition of cryoprotectant. The main differences between low- and high-resolution structures arise from correcting side-chain rotamers in the core of the protein as well as on the surface. These changes improve various structure-validation criteria.

Crystallization↗
PubMed2000

Applications of single-wavelength anomalous dispersion at high and atomic resolution.

Two examples of the application of single-wavelength anomalous dispersion (SAD) in macromolecular structure determination are described, both using the statistical phasing program SHARP. For the holmium-substituted calcium-binding protein psoriasin (22.7 kDa), a set of accurate phases has been obtained to a resolution of 1.05 A without recourse to an atomic model of the molecule. The accuracy of the phases resulted in an electron-density map of a quality comparable to sigma(A)-weighted 2mF(o) - DF(c) maps derived from the final model refined with SHELX97. Comparison of the refined and SAD electron-density maps showed significant discrepancies resulting from the iterative refinement in reciprocal space. Additionally, it is shown that the structure of psoriasin can be determined from native data extending to 2.0 A alone by exploiting the minute anomalous signal from a bound zinc ion.

Biomarkers, Tumor↗
PubMed2000

Modelling prior distributions of atoms for macromolecular refinement and completion.

Until modelling is complete, macromolecular structures are refined in the absence of a model for some of the atoms in the crystal. Techniques for defining positional probability distributions of atoms, and using them to model the missing part of a macromolecular crystal structure and the bulk solvent, are described. The starting information may consist of either a tentative structural model for the missing atoms or an electron-density map. During structure completion and refinement, the use of probability distributions enables the retention of low-resolution phase information while avoiding premature commitment to uncertain higher resolution features. Homographic exponential modelling is proposed as a flexible, compact and robust parametrization that proves to be superior to a traditional Fourier expansion in approximating a model protein envelope. The homographic exponential model also has potential applications to ab initio phasing of Fourier amplitudes associated with macromolecular envelopes.

Animals↗
PubMed2000

In-house low-resolution X-ray crystallography.

It is demonstrated that standard in-house protein crystal X-ray diffraction apparatus can be used to measure very low resolution reflections with only a few modifications. The apparatus and modifications are described in detail and tested on two different macromolecular crystal samples: lysozyme and the 30S ribosomal subunit. Contrast-variation measurements on tetragonal hen egg-white lysozyme demonstrate the potential usefulness of the apparatus in providing accurate data for the determination of macromolecular envelopes. In contrast, the measurement of very low resolution diffraction from crystals of the 30S ribosome subunit illustrates how in-house facilities can provide data from small weakly diffracting crystals of a very large macromolecule.

Animals↗
PubMed1999

Can anomalous signal of sulfur become a tool for solving protein crystal structures?

A general method for solving the phase problem from native crystals of macromolecules has long eluded structural biology. For well diffracting crystals this goal can now be achieved, as is shown here, thanks to modern data collection techniques and new statistical phasing algorithms. Using solely a native crystal of tetragonal hen egg-white lysozyme, a protein of 14 kDa molecular mass, it was possible to detect the positions of the ten sulfur and seven chlorine atoms from their anomalous signal, and proceed from there to obtain an electron-density map of very high quality.

Algorithms↗
PubMed1999

The 1.2 A crystal structure of hirustasin reveals the intrinsic flexibility of a family of highly disulphide-bridged inhibitors.

BACKGROUND: Leech-derived inhibitors have a prominent role in the development of new antithrombotic drugs, because some of them are able to block the blood coagulation cascade. Hirustasin, a serine protease inhibitor from the leech Hirudo medicinalis, binds specifically to tissue kallikrein and possesses structural similarity with antistasin, a potent factor Xa inhibitor from Haementeria officinalis. Although the 2.4 A structure of the hirustasin-kallikrein complex is known, classical methods such as molecular replacement were not successful in solving the structure of free hirustasin. RESULTS: Ab initio real/reciprocal space iteration has been used to solve the structure of free hirustasin using either 1.4 A room temperature data or 1.2 A low temperature diffraction data. The structure was also solved independently from a single pseudo-symmetric gold derivative using maximum likelihood methods. A comparison of the free and complexed structures reveals that binding to kallikrein causes a hinge-bending motion between the two hirustasin subdomains. This movement is accompanied by the isomerisation of a cis proline to the trans conformation and a movement of the P3, P4 and P5 residues so that they can interact with the cognate protease. CONCLUSIONS: The inhibitors from this protein family are fairly flexible despite being highly cross-linked by disulphide bridges. This intrinsic flexibility is necessary to adopt a conformation that is recognised by the protease and to achieve an optimal fit, such observations illustrate the pitfalls of designing inhibitors based on static lock-and-key models. This work illustrates the potential of new methods of structure solution that require less or even no prior phase information.

Amino Acid Sequence↗
PubMed1999

A multisolution method of phase determination by combined maximization of entropy and likelihood. VI. The use of error-correcting codes as a source of phase permutation and their application to the phase problem in powder, electron and macromolecular crystallography.

The use of error-correcting codes as a source of efficient designs of phase permutation schemes is described. Three codes are used, all taken from the Bricogne BUSTER program [Bricogne (1993). Acta Cryst. D49, 37-60]: the Hamming [7, 4, 3], the Nordström-Robinson (16, 256, 6) and the Golay [24, 12, 8] or its punctured [23, 12, 7] form. These are used in a maximum-entropy-likelihood phasing environment to carry out phase permutation of basis-set reflections instead of the usual quadrant permutation or magic integer approaches. The use of codes in this way inevitably introduces some errors in the phase choices, but for most structures this is not significant especially when the gain in sampling efficiency is considered. For example, the Golay [24, 14, 8] allows the permutation of 24 centric phases in such a way that only 4096 phase sets are produced instead of 2(24) = 16777216, and one of these sets has, at most, only four wrong phases. The method is successfully applied to three powder diffraction data sets of increasing complexity, and with increasing degrees of overlap {Mg(3)BN(3), Sigma-2 ([Si(64)O(128)].4C(10)H(17)N) and the NU-3 zeolite}, a sparse electron diffraction data set for buckminsterfullerene, C(60), and the small protein molecule crambin at 3 Å resolution where 42 reflections are phased with a Uweighted mean phase error of 58.5 degrees.

Journal Article↗
PubMed1998

Crystal structure of the anti-fungal target N-myristoyl transferase.

N-myristoyl transferase (NMT) catalyzes the transfer of the fatty acid myristate from myristoyl-CoA to the N-terminal glycine of substrate proteins, and is found only in eukaryotic cells. The enzyme in this study is the 451 amino acid protein produced by Candida albicans, a yeast responsible for the majority of systemic infections in immuno-compromised humans. NMT activity is essential for vegetative growth, and the structure was determined in order to assist in the discovery of a selective inhibitor of NMT which could be developed as an anti-fungal drug. NMT has no sequence homology with other protein sequences and has a novel alpha/beta fold which shows internal two-fold symmetry, which may be a result of gene duplication. On one face of the protein there is a long, curved, relatively uncharged groove, at the center of which is a deep pocket. The pocket floor is negatively charged due to the vicinity of the C-terminal carboxylate and a nearby conserved glutamic acid residue, which separates the pocket from a cavity. These observations, considered alongside the positions of residues whose mutation affects substrate binding and activity, suggest that the groove and pocket are the sites of substrate binding and the floor of the pocket is the catalytic center.

Acyl Coenzyme A↗
PubMed1997

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. The diffraction data are of excellent quality. The combination of isomorphous replacement and anomalous dispersion of a single xenon heavy-atom derivative allowed accurate phase determination and the computation of a high-quality electron density map of the protein molecule. This is the first fully documented report on a complete protein crystallography experiment, from data collection up to phase determination and calculation of an electron density map, carried out with data obtained at ultra-short wavelengths. Experimental considerations as well as possible advantages and drawbacks of protein crystallography at very short and ultra-short wavelengths are discussed.

Journal Article↗
PubMed1997

High-pressure krypton gas and statistical heavy-atom refinement: a successful combination of tools for macromolecular structure determination.

The noble gas krypton is shown to bind to crystallized proteins in a similar way to xenon [Schiltz, Prangé & Fourme (1994). J. Appl. Cryst. 27, 950-960]. Preliminary tests show that the major krypton binding sites are essentially identical to those of xenon. Noticeable substitution is achieved only at substantially higher pressures (above 50 x 10(5) Pa). As is the case for xenon, the protein complexes with krypton are highly isomorphous with the native structure so that these complexes can be used for phase determination in protein crystallography. Krypton is not as heavy as xenon, but its K-absorption edge is situated at a wavelength (0.86 A) that is readily accessible on synchrotron radiation sources. As a test case, X-ray diffraction data at the high-energy side of the K edge were collected on a crystal of porcine pancreatic elastase (molecular weight of 25.9 kDa) put under a krypton gas pressure of 56 x 10(5) Pa. The occupancy of the single Kr atom is approximately 0.5, giving isomorphous and anomalous scattering strengths of 15.2 and 1.9 e, respectively. This derivative could be used successfully for phase determination with the SIRAS method (single isomorphous replacement with anomalous scattering). After phase improvement by solvent flattening, the resulting electron-density map is of exceptionally high quality, and has a correlation coefficient of 0.85 with a map calculated from the refined native structure. Careful data collection and processing, as well as the correct statistical treatment of isomorphous and anomalous signals have proven to be crucial in the determination of this electron-density map. Heavy-atom refinement and phasing were carried out with the program SHARP, which is a fully fledged implementation of the maximum-likelihood theory for heavy-atom refinement [Bricogne (1991). Crystallographic Computing 5, edited by D. Moras, A. D. Podjarny & J. C. Thierry, pp. 257-297. Oxford: Clarendon Press]. It is concluded that the use of xenon and krypton derivatives, when they can be obtained, associated with statistical heavy-atom refinement will allow one to overcome the two major limitations of the isomorphous replacement method i.e. non-isomorphism and the problem of optimal estimation of heavy-atom parameters.

Journal Article↗
PubMed1997

Automated correlation and averaging of three-dimensional reconstructions obtained by electron tomography.

We have developed a least-squares refinement procedure that in an automated way performs three-dimensional alignment and averaging of objects from multiple reconstructions. The computer implementation aligns the three-dimensional structures by a two-step procedure that maximizes the density overlap for all objects. First, an initial average density is built by successive incorporation of individual objects, after a global search for their optimal three-dimensional orientations. Second, the initial average is subsequently refined by excluding individual objects one at a time, realigning them with the reduced average containing all other objects and including them into the average again. The refinement is repeated until no further change of the average occurs. The resulting average model is therefore minimally biased by the order in which the individual reconstructions are incorporated into the average. The performance of the procedure was tested using a synthetic data set of randomly oriented objects with Poisson-distributed noise added. The program managed well to align and average the objects at the signal/noise ratio 1.0. The increase in signal/noise ratio was in all investigated cases almost equal to the expected square root of the number of objects. The program was also successfully tested on a set of authentic three-dimensional reconstructions from an in situ specimen containing Escherichia coli 70S ribosomes, where the immediate environment of the reconstructed objects may also contain variable amounts of other structures.

Automation↗