The VIZIER project: preparedness against pathogenic RNA viruses.

Life-threatening RNA viruses emerge regularly, and often in an unpredictable manner. Yet, the very few drugs available against known RNA viruses have sometimes required decades of research for development. Can we generate preparedness for outbreaks of the, as yet, unknown viruses? The VIZIER (VIral enZymes InvolvEd in Replication) (http://www.vizier-europe.org/) project has been set-up to develop the scientific foundations for countering this challenge to society. VIZIER studies the most conserved viral enzymes (that of the replication machinery, or replicases) that constitute attractive targets for drug-design. The aim of VIZIER is to determine as many replicase crystal structures as possible from a carefully selected list of viruses in order to comprehensively cover the diversity of the RNA virus universe, and generate critical knowledge that could be efficiently utilized to jump-start research on any emerging RNA virus. VIZIER is a multidisciplinary project involving (i) bioinformatics to define functional domains, (ii) viral genomics to increase the number of characterized viral genomes and prepare defined targets, (iii) proteomics to express, purify, and characterize targets, (iv) structural biology to solve their crystal structures, and (v) pre-lead discovery to propose active scaffolds of antiviral molecules.

Tryptophanyl-tRNA synthetase crystal structure reveals an unexpected homology to tyrosyl-tRNA synthetase.

BACKGROUND: Tryptophanyl-tRNA synthetase (TrpRS) catalyzes activation of tryptophan by ATP and transfer to tRNA(Trp), ensuring translation of the genetic code for tryptophan. Interest focuses on mechanisms for specific recognition of both amino acid and tRNA substrates. RESULTS: Maximum-entropy methods enabled us to solve the TrpRS structure. Its three parts, a canonical dinucleotide-binding fold, a dimer interface, and a helical domain, have enough structural homology to tyrosyl-tRNA synthetase (TyrRS) that the two enzymes can be described as conformational isomers. Structure-based sequence alignment shows statistically significant genetic homology. Structural elements interacting with the activated amino acid, tryptophanyl-5'AMP, are almost exactly as seen in the TyrRS:tyrosyl-5'AMP complex. Unexpectedly, side chains that recognize indole are also highly conserved, and require reorientation of a 'specificity-determining' helix containing a conserved aspartate to assure selection of tryptophan versus tyrosine. The carboxy terminus, which is disordered and therefore not seen in TyrRS, forms part of the dimer interface in TrpRS. CONCLUSIONS: For the first time, the Bayesian statistical paradigm of entropy maximization and likelihood scoring has played a critical role in an X-ray structure solution. Sequence relatedness of structurally superimposable residues throughout TrpRS and TyrRS implies that they diverged more recently than most aminoacyl-tRNA synthetases. Subtle, tertiary structure changes are crucial for specific recognition of the two different amino acids. The conformational isomerism suggests that movement of the KMSKS loop, known to occur in the TyrRS transition state for amino acid activation, may provide a basis for conformational coupling during catalysis.

Crystal structure of a fungal elicitor secreted by Phytophthora cryptogea, a member of a novel class of plant necrotic proteins.

BACKGROUND: Elicitins form a novel class of plant necrotic proteins which are secreted by Phytophthora and Pythium fungi, parasites of many economically important crops. These proteins induce leaf necrosis in infected plants and elicit an incompatible hypersensitive-like reaction, leading to the development of a systemic acquired resistance against a range of fungal and bacterial plant pathogens. No crystal structures of this class of protein are available. The crystal structure determination of beta-cryptogein (CRY), secreted by Phytophthora cryptogea, was undertaken to identify structural features important for the necrotic activity of elicitins. RESULTS: The structure of CRY was determined using the multiwavelength anomalous diffraction technique and refined to 2.2 A resolution. The overall structure has a novel fold consisting of six alpha helices and a beak-like motif, whose sequence is highly conserved within the family, composed of an antiparallel two-stranded beta sheet and an omega loop. This motif is assumed to be a major recognition site for a putative receptor and/or ligand. Two other distinct binding sites seem to be correlated to the level of necrotic activity of elicitins. CONCLUSIONS: The determination of the crystal structure of a member of the elicitin family may make it possible to separate the activity that causes leaf necrosis from that inducing systemic acquired resistance to pathogens, making it feasible to engineer a non-toxic elicitin that only elicits plant defences. Such studies should aid the development of non-toxic agricultural pest control.

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.

Intramolecular dielectric screening in proteins.

This paper investigates the microscopic mechanisms of charge screening by proteins. For this purpose, we introduce the generalized susceptibility of a protein in response to a point charge, which is a scalar quantity dependent on position within the protein. The contribution to the susceptibility from atomic polarizabilities, associated with electronic degrees of freedom, is found to be highly uniform. By contrast, that from dynamic dipolar relaxation, associated with nuclear degrees of freedom, varies greatly between different regions of the protein. We investigate the possible rôle of this variation in the activity of proteins that interact functionally with charged species, and we formulate and test the hypothesis that this variation is correlated to functional activity. Model calculations give encouraging support to this hypothesis. The protein's dielectric properties are represented by a standard model in which electronic relaxation is described by a set of atomic polarizabilities, and dipolar relaxation is treated as a perturbation to normal mode dynamics. The model yields the desired susceptibility in closed form. Its obvious limitations are discussed. It is applied to several test systems, and is compared to various continuum models. Four model alpha-helices are considered, three of which play a rôle in vivo in the binding of charged ligands. We show that the intramolecular screening, and its spatial variation, can indeed play a part in this binding. The electron transfer between ferri- and ferrocytochrome c is considered. The dielectric relaxation of each molecule, associated respectively with its oxidation or its reduction, is known to be directly related to the activation free energy for the electron transfer reaction. Our analysis of the dielectric susceptibility will thus permit an estimate of this activation free energy. We show that the relaxation of the atomic positions ("dipolar relaxation") contributes 1 kcal/mol to this activation free energy, and that the molecule achieves this low value by providing a low dipolar susceptibility throughout its central part. In this case, the spatial variation of the susceptibility has a clear functional rôle.

Structure of tomato busy stunt virus IV. The virus particle at 2.9 A resolution.

The structure of tomato bushy stunt virus has been determined crystallographically to 2.9 A resolution. Details are presented of both the molecular structure and the methods by which it has been solved. The icosahedrally symmetric viral shell is composed of 180 protein subunits (Mr 43,000), with three similar but distinct modes of subunit bonding. This capacity for alternative packing is due to localized flexibility in the folded polypeptide (hinges between domains) and to multiple conformations for surface side-chains. The polypeptide backbone has an essentially invariant fold within a compact domain. A mechanism for correct positioning of the different modes of subunit interaction is evident from the structure of the TBSV particle. Thirty-five residues of the polypeptide chain fold in an ordered way on 60 of the 180 subunits, forming an internal framework. Interaction of folded domains with this framework permits accuracy of long-range geometry (correct curvature and closure) to be determined by unambiguous switching between alternative local contact angles. RNA packs tightly into the particle interior. Protein-RNA interactions occur through parts of the subunit that are flexibly linked to the well-ordered domains of the shell. This variable interaction imposes minimum restrictions on the folding of the RNA chain.

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.

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.

A multisolution method of phase determination by combined maximization of entropy and likelihood. V. The use of likelihood as a discriminator of phase sets produced by the SAYTAN program for a small protein.

The use of a likelihood criterion associated with maximum-entropy (ME) extrapolation for selecting phase sets as part of a new multisolution phasing strategy, already applied to solving small crystal structures from single-crystal data [Gilmore, Bricogne & Bannister (1990). Acta Cryst. A46, 297-308] and X-ray powder diffraction data [Gilmore, Henderson & Bricogne (1991). Acta Cryst. A47, 830-841], has been tested on the small protein avian pancreatic polypeptide (APP) with 301 non-H atoms in the asymmetric unit in space group C2. A collection of 50 phase sets for APP were provided by Woolfson & Yao. They had been generated from random starting phases by the SAYTAN procedure [Woolfson & Yao (1990). Acta Cryst. A46, 409-413] using data to a resolution of 0.98 A. Six of these had an unweighted mean absolute phase error, mean value of magnitude of delta phi, of less than 50 degrees, the remainder having phase errors of 60 degrees or more. However, none of the conventional figures of merit were able to identify these preferred sets. Each phase set was subjected to our standard procedure of entropy maximization and of evaluation of the log-likelihood gain resulting from the associated ME extrapolation. With only a small subset of data (to 2 A resolution), the likelihood criterion identified unambiguously the phase sets with mean value of magnitude of delta phi less than 50 degrees. In contrast, conventional figures of merit showed no such ability.(ABSTRACT TRUNCATED AT 250 WORDS)

Direct phase determination for the molecular envelope of tryptophanyl-tRNA synthetase from Bacillus stearothermophilus by X-ray contrast variation.

Monoclinic crystals of Bacillus stearothermophilus tryptophanyl-tRNA synthetase grown in the presence of substrate tryptophan (space group P2(1)) display evidence of a low-resolution trigonal space group (P321). The origin and averaging transformations for the local 32 point group of this unusually clear sixfold non-crystallographic symmetry may be inferred without prior estimation of the electron density. This local symmetry was exploited in conjunction with solvent density contrast variation to determine the shape of the molecular envelope. X-ray intensities measured from crystals equilibrated in mother liquors of three different electron densities were used to estimate three parameters for each reflection: the modulus of the envelope transform, [Gh]; and components, Xh and Yh, relative to Gh, of the structure-factor vector for the transform of intramolecular density fluctuations. The moduli ([Gh]) behave somewhat like structure-factor amplitudes from small-molecule crystals, and estimation of their unknown phases was successfully carried out by statistical direct methods. Reflections to 18 A resolution, which obey rather well the symmetry of space group P321, were merged to produce an asymmetric unit in that space group. [Gh] values for the 34 strongest of these were phased using the small-molecule direct-methods package MITHRIL [Gilmore (1984). J. Appl Cryst. 17, 42-46]. The best phase set was expanded back to the P2(1) lattice and negative density was truncated to generate initial phases for all reflections to 18 A resolution. Phase refinement by iterative imposition of the local 32 symmetry produced an envelope with convincing features consistent with known properties of the enzyme. The envelope implies that the tryptophanyl-tRNA synthetase dimer is an elongated structure with an axial ratio of about 4:1, in which the monomers have two distinct domains of unequal size. The smaller of these occurs at the dimer interface, and resembles the nucleotide binding portion of the tyrosyl-tRNA synthetase. It may therefore contain the amino-terminal one hundred or so residues, including all three cysteines, previously suggested to comprise a nucleotide-binding domain in the tryptophanyl enzyme. A purely crystallographic test of the overall features of this envelope was carried out by transporting it to a tetragonal crystal form of the same protein in which the asymmetric unit is a monomer. The small domain fits snugly inside three mercury and one gold heavy-atom binding sites for this crystal form; and symmetry-related molecules provide excellent, but very different, lattice contacts in nearly all directions.

Overcoming non-isomorphism by phase permutation and likelihood scoring: solution of the TrpRS crystal structure.

Entropy maximization to maximum likelihood, constrained jointly by the best available experimental phases and by a sufficiently good envelope, can bring about substantial model-independent map improvement, even at medium (3.1 A) resolution [Xiang, Carter, Bricogne & Gilmore (1993). Acta Cryst. D49, 193-212]. In the crystal structure determination of the Bacillus stearothermophilus tryptophanyl-tRNA synthetase (TrpRS), however, the following had to be dealt with simultaneously: (1) a serious lack of isomorphism in the heavy-atom derivatives, resulting in large starting-phase errors; and (2) an initially poorly known molecular envelope. Because the constraints--both phases and envelope--were insufficiently well determined at the outset, maximum-entropy solvent flattening as previously applied was unsuccessful. Rather than improving the maps, it led to a deterioration of their quality, accompanied by a dramatic decrease of the log-likelihood gain as phases were extended from about 5 A resolution to the 2.9 A limit of the diffraction data. This deadlock was broken by the identification of strong reflections, which were initially unphased and which were inaccessible by maximum-entropy extrapolation from the phased ones, and by permutation of the phases of these reflections so as to sample the space of possible electron-density and envelope modifications they represented. Permutation was carried out by successive full and incomplete factorial designs [Carter & Carter (1979). J. Biol. Chem. 254, 12219-12223] for 28 strong reflections selected in decreasing order of their 'renormalized' structure-factor amplitudes. The permuted reflections included one reflection for which the probability distribution from multiple isomorphous replacement with anomalous scattering (MIRAS) indicated an incorrect phase with a high figure of merit and which consequently had a large renormalized structure factor. A similar permutation was carried out for six different binary choices related to the calculation and description of the molecular envelope. Permutation experiments were scored using the log-likelihood gain and contrasts for each main effect were analyzed by multiple-regression least squares. Student t tests provided significant and reliable indications for a large majority of the permuted reflections and for all six hypotheses related to the molecular envelope. The resulting phase improvement made it possible to assign positions (hitherto unobtainable) for nine of the ten selenium atoms in an isomorphous difference Fourier map for selenomethionine-substituted TrpRS crystals and hence to solve the structure. Phase-permutation methods continued to be useful in producing improved maps from all the available isomorphous-replacement phase information and therefore played a critical role in solving the structure.(ABSTRACT TRUNCATED AT 400 WORDS)

Intermolecular versus intramolecular interactions of the vinculin binding site 33 of talin.

The cytoskeletal proteins talin and vinculin are localized at cell-matrix junctions and are key regulators of cell signaling, adhesion, and migration. Talin couples integrins via its FERM domain to F-actin and is an important regulator of integrin activation and clustering. The 220 kDa talin rod domain comprises several four- and five-helix bundles that harbor amphipathic α-helical vinculin binding sites (VBSs). In its inactive state, the hydrophobic VBS residues involved in binding to vinculin are buried within these helix bundles, and the mechanical force emanating from bound integrin receptors is thought necessary for their release and binding to vinculin. The crystal structure of a four-helix bundle of talin that harbors one of these VBSs, coined VBS33, was recently determined. Here we report the crystal structure of VBS33 in complex with vinculin at 2 Å resolution. Notably, comparison of the apo and vinculin bound structures shows that intermolecular interactions of the VBS33 α-helix with vinculin are more extensive than the intramolecular interactions of the VBS33 within the talin four-helix bundle.

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.

Direct phase determination by entropy maximization and likelihood ranking: status report and perspectives.

A new multisolution phasing method based on entropy maximization and likelihood ranking, proposed for the specific purpose of extending probabilistic direct methods to the field of macromolecules, has been implemented in two different computer programs and applied to a wide variety of problems. The latter comprise the determination of small crystal structures from X-ray diffraction data obtained from single crystals or from powders, and from electron diffraction data partially phased by image processing of electron micrographs, the ab initio generation and ranking of phase sets for small proteins; and the improvement of poor quality phases for a larger protein at medium resolution under constraint of solvent flatness. These applications show that the primary goal of this new method - namely increasing the accuracy and sensitivity of probabilistic phase indications compared with conventional direct methods - has been achieved. The main components of the method are (1) a tree-directed search through a space of trial phase sets; (2) the saddle-point method for calculating joint probabilities of structure factors, using entropy maximization; (3) likelihood-based scores to rank trial phase sets and prune the search tree; (4) efficient schemes, based on error-correcting codes, for sampling trial phase sets; (5) a statistical analysis of the scores for automatically selecting reliable phase indications. They have been implemented to varying degrees of completeness in a computer program (BUSTER) and tested on two small structures as well as on the small protein crambin. The main obstructions to successful ab initio phasing in the latter case seem to reside in the accumulation of phase sampling errors and in the lack of a properly defined molecular envelope, both of which can be remedied within the methods proposed. A review of the Bayesian statistical theory encompassing all phasing procedures, proposed earlier as an extension of the initial theory, shows that the techniques now available in BUSTER bring closer a number of major enhancements of standard macromolecular phasing techniques, namely isomorphous replacement, molecular replacement, solvent flattening and non-crystallographic symmetry averaging. The gradual implementation of the successive stages of this 'Bayesian programme' should lead to an increasingly integrated, effective and dependable phasing procedure for macromolecular structure determination.

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.

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.

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.

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.