The majority of previously deposited X-ray structures can be improved by applying current refinement methods.
Structural biology, homology modelling and rational drug design require accurate three-dimensional macromolecular coordinates. However, the coordinates in the Protein Data Bank (PDB) have not all been obtained using the latest experimental and computational methods. In this study a method is presented for automated re-refinement of existing structure models in the PDB. A large-scale benchmark with 16 807 PDB entries showed that they can be improved in terms of fit to the deposited experimental X-ray data as well as in terms of geometric quality. The re-refinement protocol uses TLS models to describe concerted atom movement. The resulting structure models are made available through the PDB_REDO databank (http://www.cmbi.ru.nl/pdb_redo/). Grid computing techniques were used to overcome the computational requirements of this endeavour.
X-ray crystallography; refinement; structure validation; Protein Data Bank; grid computing
The ability of pathogenesis-related proteins of family 10 to bind a broad spectrum of ligands is considered to play a key role for their physiological and pathological functions. In particular, Bet v 1, an archetypical allergen from birch pollen, is described as a highly promiscuous ligand acceptor. However, the detailed recognition mechanisms, including specificity factors discriminating binding properties of naturally occurring Bet v 1 variants, are poorly understood.
Here, we report crystal structures of Bet v 1 variants in complex with an array of ligands at a resolution of up to 1.2 Å. Residue 30 within the hydrophobic pocket not only discriminates in high and low IgE binding Bet v 1 isoforms but also induces a drastic change in the binding mode of the model ligand deoxycholate. Ternary crystal structure complexes of Bet v 1 with several ligands together with the fluorogenic reporter 1-anilino-8-naphthalene sulfonate explain anomalous fluorescence binding curves obtained from 1-anilino-8-naphthalene sulfonate displacement assays. The structures reveal key interaction residues such as Tyr83 and rationalize both the binding specificity and promiscuity of the so-called hydrophobic pocket in Bet v 1.
The intermolecular interactions of Bet v 1 reveal an unexpected complexity that will be indispensable to fully understand its roles within the physiological and allergenic context.
► Ligand binding to Bet v 1 may contribute to explain its allergenicity. ► High-resolution structures reveal the binding mode of diverse ligands to Bet v 1. ► Residue 30 starkly influences the binding properties of different Bet v 1 isoforms. ► Ternary complexes with diverse ligands explain anomalous fluorescence binding curves. ► Betv1 isoforms differ in ligand binding, which may translate into their allergenicity.
ANS, 1-anilino-8-naphthalene sulfonate; BRA, brassinolide; DXC, deoxycholate; iDXC, inner deoxycholate; oDXC, outer deoxycholate; LPS, lipopolysaccharide; MPD, 2-methyl-2,4-pentanediol; NDSB-256, non-detergent sulfobetaine 256; PR-10, pathogenesis-related protein 10; PDB, Protein Data Bank; molecular allergenicity; ANS displacement assay; structure–allergenicity relationship; binding specificity and promiscuity; dressed allergens
The major birch pollen allergen, Bet v 1, is a member of the ubiquitous PR-10 family of plant pathogenesis-related proteins. In recent years, a number of diverse plant proteins with low sequence similarity to Bet v 1 was identified. In addition, determination of the Bet v 1 structure revealed the existence of a large superfamily of structurally related proteins. In this study, we aimed to identify and classify all Bet v 1-related structures from the Protein Data Bank and all Bet v 1-related sequences from the Uniprot database.
Structural comparisons of representative members of already known protein families structurally related to Bet v 1 with all entries of the Protein Data Bank yielded 47 structures with non-identical sequences. They were classified into eleven families, five of which were newly identified and not included in the Structural Classification of Proteins database release 1.71. The taxonomic distribution of these families extracted from the Pfam protein family database showed that members of the polyketide cyclase family and the activator of Hsp90 ATPase homologue 1 family were distributed among all three superkingdoms, while members of some bacterial families were confined to a small number of species. Comparison of ligand binding activities of Bet v 1-like superfamily members revealed that their functions were related to binding and metabolism of large, hydrophobic compounds such as lipids, hormones, and antibiotics. Phylogenetic relationships within the Bet v 1 family, defined as the group of proteins with significant sequence similarity to Bet v 1, were determined by aligning 264 Bet v 1-related sequences. A distance-based phylogenetic tree yielded a classification into 11 subfamilies, nine exclusively containing plant sequences and two subfamilies of bacterial proteins. Plant sequences included the pathogenesis-related proteins 10, the major latex proteins/ripening-related proteins subfamily, and polyketide cyclase-like sequences.
The ubiquitous distribution of Bet v 1-related proteins among all superkingdoms suggests that a Bet v 1-like protein was already present in the last universal common ancestor. During evolution, this protein diversified into numerous families with low sequence similarity but with a common fold that succeeded as a versatile scaffold for binding of bulky ligands.
An evaluation of validation and real-space intervention possibilities for improving existing automated (re-)refinement methods.
The deposition of X-ray data along with the customary structural models defining PDB entries makes it possible to apply large-scale re-refinement protocols to these entries, thus giving users the benefit of improvements in X-ray methods that have occurred since the structure was deposited. Automated gradient refinement is an effective method to achieve this goal, but real-space intervention is most often required in order to adequately address problems detected by structure-validation software. In order to improve the existing protocol, automated re-refinement was combined with structure validation and difference-density peak analysis to produce a catalogue of problems in PDB entries that are amenable to automatic correction. It is shown that re-refinement can be effective in producing improvements, which are often associated with the systematic use of the TLS parameterization of B factors, even for relatively new and high-resolution PDB entries, while the accompanying manual or semi-manual map analysis and fitting steps show good prospects for eventual automation. It is proposed that the potential for simultaneous improvements in methods and in re-refinement results be further encouraged by broadening the scope of depositions to include refinement metadata and ultimately primary rather than reduced X-ray data.
The decision-making algorithms and software used in PDB_REDO to re-refine and rebuild crystallographic protein structures in the PDB are presented and discussed.
Developments of the PDB_REDO procedure that combine re-refinement and rebuilding within a unique decision-making framework to improve structures in the PDB are presented. PDB_REDO uses a variety of existing and custom-built software modules to choose an optimal refinement protocol (e.g. anisotropic, isotropic or overall B-factor refinement, TLS model) and to optimize the geometry versus data-refinement weights. Next, it proceeds to rebuild side chains and peptide planes before a final optimization round. PDB_REDO works fully automatically without the need for intervention by a crystallographic expert. The pipeline was tested on 12 000 PDB entries and the great majority of the test cases improved both in terms of crystallographic criteria such as R
free and in terms of widely accepted geometric validation criteria. It is concluded that PDB_REDO is useful to update the otherwise ‘static’ structures in the PDB to modern crystallographic standards. The publically available PDB_REDO database provides better model statistics and contributes to better refinement and validation targets.
validation; refinement; model building; automation; PDB
The application of a multivariate likelihood function to a single isomorphous replacement with anomalous scattering experiment improves phasing and automated model building with iterative refinement in the test cases shown.
A likelihood function based on the multivariate probability distribution of all observed structure-factor amplitudes from a single isomorphous replacement with anomalous scattering experiment has been derived and implemented for use in substructure refinement and phasing as well as macromolecular model refinement. Efficient calculation of a multidimensional integration required for function evaluation has been achieved by approximations based on the function’s properties. The use of the function in both phasing and protein model building with iterative refinement was essential for successful automated model building in the test cases presented.
multivariate normal probability distribution; single isomorphous replacement with anomalous scattering; experimental phasing; direct incorporation of prior phase information
A fast analytical method for calculating mask-based bulk-solvent scale factors and overall anisotropic correction factors is introduced.
A fast and robust method for determining the parameters for a flat (mask-based) bulk-solvent model and overall scaling in macromolecular crystallographic structure refinement and other related calculations is described. This method uses analytical expressions for the determination of optimal values for various scale factors. The new approach was tested using nearly all entries in the PDB for which experimental structure factors are available. In general, the resulting R factors are improved compared with previously implemented approaches. In addition, the new procedure is two orders of magnitude faster, which has a significant impact on the overall runtime of refinement and other applications. An alternative function is also proposed for scaling the bulk-solvent model and it is shown that it outperforms the conventional exponential function. Similarly, alternative methods are presented for anisotropic scaling and their performance is analyzed. All methods are implemented in the Computational Crystallography Toolbox (cctbx) and are used in PHENIX programs.
bulk solvent; scaling; anisotropy; structure refinement; PHENIX
Up to 70% of birch pollen-allergic individuals show adverse reactions to certain plant foods. This cross-reactivity is caused by sensitisation to the major birch pollen allergen Bet v 1 and binding of Bet v 1-specific IgE antibodies to homologous plant food allergens. We aimed to assess the importance of selected conformational epitopes for IgE binding to Bet v 1.
Chimeras of Bet v 1.0101 and its homologue Api g 1.0101 were constructed. In each of the 4 chimeras, roughly one fourth of the surface residues of Api g 1.0101 were replaced by corresponding residues of Bet v 1.0101. The proteins were expressed in Escherichia coli and purified by chromatographic methods. Secondary structures were checked by CD-spectroscopy. IgE ELISA with Bet v 1.0101, Api g 1.0101 and the chimeras were performed with sera of 63 Bet v 1-sensitized birch pollen allergic patients. For inhibition ELISAs, chimeras were coated and inhibition was performed with the chimeras or Api g 1.0101.
IgE binding to Api g 1.0101, Api-Bet-1, -2, -3 and -4 was observed for 22, 81, 79, 70 and 38% of the sera, respectively. To assess the relevance of the grafted regions for IgE binding to Bet v 1, the amounts of IgE binding to the chimeras were compared with those to Api g 1.0101. Most of the sera recognised either 3 chimeras (39%) or all 4 chimeras (21%) better than Api g 1.0101. Only a minority of the sera showed increased binding to a single chimera. Inhibition ELISAs confirmed the presence of IgE specific for the grafted regions.
Our study indicates that the epitope recognition profile of Bet v 1-specific IgE is highly patient specific. Due to the different IgE binding patterns to Bet v 1, determined by binding of IgE to different chimeras, the existence of a single major IgE epitope on Bet v 1 can be excluded. Moreover, the Bet v 1-specific IgE repertoire is polyclonal and the IgE epitopes are distributed over the whole surface of Bet v 1.
Among birch pollen allergic patients up to 70% develop allergic reactions to Bet v 1-homologue food allergens such as Api g 1 (celery) or Dau c 1 (carrot), termed as birch pollen-related food allergy. In most cases, specific immunotherapy with birch pollen extracts does not reduce allergic symptoms to the homologue food allergens. We therefore genetically engineered a multi-allergen chimer and tested if mucosal treatment with this construct could represent a novel approach for prevention of birch pollen-related food allergy.
BALB/c mice were poly-sensitized with a mixture of Bet v 1, Api g 1 and Dau c 1 followed by a sublingual challenge with carrot, celery and birch pollen extracts. For prevention of allergy sensitization an allergen chimer composed of immunodominant T cell epitopes of Api g 1 and Dau c 1 linked to the whole Bet v 1 allergen, was intranasally applied prior to sensitization.
Intranasal pretreatment with the allergen chimer led to significantly decreased antigen-specific IgE-dependent β-hexosaminidase release, but enhanced allergen-specific IgG2a and IgA antibodies. Accordingly, IL-4 levels in spleen cell cultures and IL-5 levels in restimulated spleen and cervical lymph node cell cultures were markedly reduced, while IFN-γ levels were increased. Immunomodulation was associated with increased IL-10, TGF-β and Foxp3 mRNA levels in NALT and Foxp3 in oral mucosal tissues. Treatment with anti-TGF-β, anti-IL10R or anti-CD25 antibodies abrogated the suppression of allergic responses induced by the chimer.
Our results indicate that mucosal application of the allergen chimer led to decreased Th2 immune responses against Bet v 1 and its homologue food allergens Api g 1 and Dau c 1 by regulatory and Th1-biased immune responses. These data suggest that mucosal treatment with a multi-allergen vaccine could be a promising treatment strategy to prevent birch pollen-related food allergy.
Synthetic contiguous overlapping peptides (COPs) may represent an alternative to allergen extracts or recombinant allergens for allergen specific immunotherapy. In combination, COPs encompass the entire allergen sequence, providing all potential T cell epitopes, while preventing IgE conformational epitopes of the native allergen.
Individual COPs were derived from the sequence of Bet v 1, the major allergen of birch pollen, and its known crystal structure, and designed to avoid IgE binding. Three sets of COPs were tested in vitro in competition ELISA and basophil degranulation assays. Their in vivo reactivity was determined by intraperitoneal challenge in rBet v 1 sensitized mice as well as by skin prick tests in volunteers with allergic rhinoconjunctivitis to birch pollen.
The combination, named AllerT, of three COPs selected for undetectable IgE binding in competition assays and for the absence of basophil activation in vitro was unable to induce anaphylaxis in sensitized mice in contrast to rBet v 1. In addition no positive reactivity to AllerT was observed in skin prick tests in human volunteers allergic to birch pollen. In contrast, a second set of COPs, AllerT4-T5 displayed some residual IgE binding in competition ELISA and a weak subliminal reactivity to skin prick testing.
The hypoallergenicity of contiguous overlapping peptides was confirmed by low, if any, IgE binding activity in vitro, by the absence of basophil activation and the absence of in vivo induction of allergic reactions in mouse and human.
IgE; Peptides; Immunotherapy; Birch; Pollen
We dissected the T cell activation potency and the immunoglobulin (Ig) E-binding properties (allergenicity) of nine isoforms of Bet v 1 (Bet v 1a-Bet v 1l), the major birch pollen allergen. Immunoblot experiments showed that Bet v 1 isoforms differ in their ability to bind IgE from birch pollen-allergic patients. All patients tested displayed similar IgE-binding patterns toward each particular isoform. Based on these experiments, we grouped Bet v 1 isoforms in three classes: molecules with high IgE-binding activity (isoforms a, e, and j), intermediate IgE- binding (isoforms b, c, and f), and low/no IgE-binding activity (isoforms d, g, and 1). Bet v 1a, a recombinant isoform selected from a cDNA expression library using IgE immunoscreening exhibited the highest IgE-binding activity. Isoforms a, b, d, e, and 1 were chosen as representatives from the three classes for experimentation. The potency of each isoallergen to activate T lymphocytes from birch pollen- allergic patients was assayed using peripheral blood mononuclear cells, allergen-specific T cell lines, and peptide-mapped allergen-specific T cell clones. Among the patients, some displayed a broad range of T cell- recognition patterns for Bet v 1 isoforms whereas others seemed to be restricted to particular isoforms. In spite of this variability, the highest scores for T cell proliferative responses were observed with isoform d (low IgE binder), followed by b, 1, e, and a. In vivo (skin prick) tests showed that the potency of isoforms d and 1 to induce typical urticarial type 1 reactions in Bet v 1-allergic individuals was significantly lower than for isoforms a, b, and e. Taken together, our results indicate that hypoallergenic Bet v 1 isoforms are potent activators of allergen-specific T lymphocytes, and Bet v 1 isoforms with high in vitro IgE-binding activity and in vivo allergenicity can display low T cell antigenicity. Based on these findings, we propose a novel approach for immunotherapy of type I allergies: a treatment with high doses of hypoallergenic isoforms or recombinant variants of atopic allergens. We proceed on the assumption that this measure would modulate the quality of the T helper cell response to allergens in vivo. The therapy form would additionally implicate a reduced risk of anaphylactic side effects.
Allergic reactions towards the birch major pollen allergen Bet v 1 are among the most common causes of spring pollinosis in the temperate climate zone of the Northern hemisphere. Natural Bet v 1 is composed of a complex mixture of different isoforms. Detailed analysis of recombinant Bet v 1 isoforms revealed striking differences in immunologic as well as allergenic properties of the molecules, leading to a classification of Bet v 1 isoforms into high, medium, and low IgE binding proteins. Especially low IgE binding Bet v 1 isoforms have been described as ideal candidates for desensitizing allergic patients with allergen specific immunotherapy (SIT). Since diagnosis and therapy of allergic diseases are highly dependent on recombinant proteins, continuous improvement of protein production is an absolute necessity.
Therefore, two different methods for recombinant production of a low IgE binding Bet v 1 isoform were applied; one based on published protocols, the other by implementing latest innovations in protein production. Both batches of Bet v 1.0401 were extensively characterized by an array of physicochemical as well as immunological methods to compare protein primary structure, purity, quantity, folding, aggregation state, thermal stability, and antibody binding capacity.
The experiments demonstrated that IgE antibody binding properties of recombinant isoallergens can be significantly influenced by the production method directly affecting possible clinical applications of the molecules.
The major birch pollen allergen, Betv1 of Betula verrucosa is the main causative agent of birch pollen allergy in humans. Betv1 is capable of
binding several physiological ligands including fatty acids, flavones, cytokinins and sterols. Until now, no structural information from
crystallography or NMR is available regarding binding mode of any of these ligands into the binding pocket of Betv1. In the present study thirteen
ligands have been successfully docked into the hydrophobic cavity of Betv1 and binding free energies of the complexes have been calculated using
AutoDock 3.0.5. A linear relationship with correlation coefficient (R2) of 0.6 is obtained between ΔGbs values plotted against their corresponding
IC50 values. The complex formed between Betv1 and the best docking pose for each ligand has been optimized by molecular dynamics simulation.
Here, we describe the ligand binding of Betv1, which provides insight into the biological function of this protein. This knowledge is required for
structural alteration or inhibition of some of these ligands in order to modify the allergenic properties of this protein.
Betula verrucosa; Birch pollen allergy; Docking; Molecular dynamics simulation
The Protein Data Bank (PDB) is the world-wide repository of macromolecular structure information. We present a series of databases that run parallel to the PDB. Each database holds one entry, if possible, for each PDB entry. DSSP holds the secondary structure of the proteins. PDBREPORT holds reports on the structure quality and lists errors. HSSP holds a multiple sequence alignment for all proteins. The PDBFINDER holds easy to parse summaries of the PDB file content, augmented with essentials from the other systems. PDB_REDO holds re-refined, and often improved, copies of all structures solved by X-ray. WHY_NOT summarizes why certain files could not be produced. All these systems are updated weekly. The data sets can be used for the analysis of properties of protein structures in areas ranging from structural genomics, to cancer biology and protein design.
Patients with the birch pollen allergy frequently develop hypersensitive reactions to certain plant food. These reactions result from the similarity of allergen proteins structure, which are sometimes unbound phylogenetically. The aim of this study was to investigate the diagnostic value of immunoblotting method for patients with pollinosis.
Fifty eight patients were included in the study. The clinical history: the positive result of the skin prick test with the birch extract and symptoms after consumption plant food were the condition for qualifications. The immunoblotting was performed for the patients with the positive value of birch, apple, celery and/or carrot specific IgE to confirm the cross-reactivity.
Sera of 13 patients (18 patients were analyzed) revealed positive results in the immunoblotting method. Sera of only 12 patients revealed the reaction against the birch pollen protein with a molecular weight 17 to 18 kDa corresponding to the main birch allergen Bet v 1. Sera of only 2 of these patients revealed the presence of antibodies cross-reacting with the apple protein with the same molecular weight, which may indicate the main allergens of these foods – Mal d 1. Serum of 6 patients revealed the presence of antibodies cross-reacting with apple and celery protein with the same molecular weight, which may indicate the main allergens of these foods – Mal d 1 and Api g 1. Serum of only one patient revealed the presence of antibodies cross-reacting with the apple, celery and carrot protein with the same molecular weight, which may correspond the main allergens of these foods – Mal d 1, Api g 1 Dau c 1. Additionally sera of 6 persons demonstrated the presence of antibodies reacting with apple protein with the molecular weight 10 kDa which may correspond to the lipid transfer protein (LTP). Among some of the patients, antibodies which have not been identified so far reacted with birch, apple and celery proteins.
Although the immunoblotting is an effective method confirming the existences of the cross-reactivity, it still remains the method of verifying and supplementing other diagnostic tests, and a negative result doesn't exclude the existence of this kind of allergy.
This paper describes a Python script that may be used to gather all required structure-annotation information into an mmCIF file for upload through the RCSB PDB ADIT structure-deposition interface.
Almost all successful protein structure-determination projects in the public sector culminate in a structure deposition to the Protein Data Bank (PDB). In order to expedite the deposition proces, Deposit3D has been developed. This command-line script calculates or gathers all the required structure-deposition information and outputs this data into a mmCIF file for subsequent upload through the RCSB PDB ADIT interface. Deposit3D might be particularly useful for structural genomics pipeline projects because it allows workers involved with various stages of a structure-determination project to pool their different categories of annotation information before starting a deposition session.
protein crystallography; mmCIF; structure annotation; Python
Information obtained from Nuclear Magnetic Resonance (NMR) experiments is encoded as a set of constraint lists when calculating three-dimensional structures for a protein. With the amount of constraint data from the world wide Protein Data Bank (wwPDB) that is now available, it is possible to do a global, large-scale analysis using only information from the constraints, without taking the coordinate information into account. This article describes such an analysis of distance constraints from NOE data based on a set of 1834 NMR PDB entries containing 1909 protein chains. In order to best represent the quality and extent of the data that is currently deposited at the wwPDB, only the original data as deposited by the authors was used, and no attempt was made to ‘clean up’ and further interpret this information. Because the constraint lists provide a single set of data, and not an ensemble of structural solutions, they are easier to analyse and provide a reduced form of structural information that is relevant for NMR analysis only. The online resource resulting from this analysis (http://www.ebi.ac.uk/msd/srv/docs/NMR/analysis/results/html/comparison.html) makes it possible to check, for example, how often a particular contact occurs when assigning NOESY spectra, or to find out whether a particular sequence fragment is likely to be difficult to assign. In this respect it formalises information that scientists with experience in spectrum analysis are aware of but cannot necessarily quantify. The analysis described here illustrates the importance of depositing constraints (and all other possible NMR derived information) along with the structure coordinates, as this type of information can greatly assist the NMR community.
Constraint analysis; NOE assignment; NOE distances; Nuclear Magnetic Resonance (NMR); PDB
IgE antibody-mediated allergies affect more than 25% of the population worldwide. To investigate therapeutic and preventive effects of passive immunization with allergen-specific IgG antibodies on allergy in mouse models we used clinically relevant pollen allergens. In a treatment model, mice were sensitized to the major birch pollen allergen Bet v 1 and to the major grass pollen allergens, Phl p 1 and Phl p 5 and then received passive immunization with rabbit IgG antibodies specific for the sensitizing or an unrelated allergen. In a prevention model, mice obtained passive immunization with allergen-specific rabbit IgG before sensitization. Kinetics of the levels of administered IgG antibodies, effects of administered allergen-specific IgG on allergen-specific IgE reactivity, the development of IgE and IgG responses and on immediate allergic reactions were studied by ELISA, rat basophil leukaemia degranulation assays and skin testing, respectively. Treated mice showed an approximately 80% reduction of allergen-specific IgE binding and basophil degranulation which was associated with the levels of administered allergen-specific IgG antibodies. Preventive administration of allergen-specific IgG antibodies suppressed the development of allergen-specific IgE and IgG1 antibody responses as well as allergen-induced basophil degranulation and skin reactivity. Our results show that passive immunization with allergen-specific IgG antibodies is effective for treatment and prevention of allergy to clinically important pollen allergens in a mouse model and thus may pave the road for the clinical application of allergen-specific antibodies in humans.
i.n., intranasal; SIT, allergen-specific immunotherapy; Allergy; Prevention; Treatment; Antibodies; Mouse; Passive immunization
A new data model for PDB entries of viruses and other biological assemblies with regular noncrystallographic symmetry is described.
A new scheme has been devised to represent viruses and other biological assemblies with regular noncrystallographic symmetry in the Protein Data Bank (PDB). The scheme describes existing and anticipated PDB entries of this type using generalized descriptions of deposited and experimental coordinate frames, symmetry and frame transformations. A simplified notation has been adopted to express the symmetry generation of assemblies from deposited coordinates and matrix operations describing the required point, helical or crystallographic symmetry. Complete correct information for building full assemblies, subassemblies and crystal asymmetric units of all virus entries is now available in the remediated PDB archive.
virus structures; Protein Data Bank; database integration; uniform curation; point symmetry; helical symmetry; biological assemblies
Many Protein Data Bank (PDB) users assume that the deposited structural models are of high quality but forget that these models are derived from the interpretation of experimental data. The accuracy of atom coordinates is not homogeneous between models or throughout the same model. To avoid basing a research project on a flawed model, we present a tool for assessing the quality of ligands and binding sites in crystallographic models from the PDB.
The Validation HElper for LIgands and Binding Sites (VHELIBS) is software that aims to ease the validation of binding site and ligand coordinates for non-crystallographers (i.e., users with little or no crystallography knowledge). Using a convenient graphical user interface, it allows one to check how ligand and binding site coordinates fit to the electron density map. VHELIBS can use models from either the PDB or the PDB_REDO databank of re-refined and re-built crystallographic models. The user can specify threshold values for a series of properties related to the fit of coordinates to electron density (Real Space R, Real Space Correlation Coefficient and average occupancy are used by default). VHELIBS will automatically classify residues and ligands as Good, Dubious or Bad based on the specified limits. The user is also able to visually check the quality of the fit of residues and ligands to the electron density map and reclassify them if needed.
VHELIBS allows inexperienced users to examine the binding site and the ligand coordinates in relation to the experimental data. This is an important step to evaluate models for their fitness for drug discovery purposes such as structure-based pharmacophore development and protein-ligand docking experiments.
Electron density map; Binding site structure validation; Ligand structure validation; Protein structure validation; PDB; PDB_REDO
Bet v 1 is an important cause of hay fever in northern Europe. Bet v 1 isoforms from the European white birch (Betula pendula) have been investigated extensively, but the allergenic potency of other birch species is unknown. The presence of Bet v 1 and closely related PR-10 genes in the genome was established by amplification and sequencing of alleles from eight birch species that represent the four subgenera within the genus Betula. Q-TOF LC-MSE was applied to identify which PR-10/Bet v 1 genes are actually expressed in pollen and to determine the relative abundances of individual isoforms in the pollen proteome.
All examined birch species contained several PR-10 genes. In total, 134 unique sequences were recovered. Sequences were attributed to different genes or pseudogenes that were, in turn, ordered into seven subfamilies. Five subfamilies were common to all birch species. Genes of two subfamilies were expressed in pollen, while each birch species expressed a mixture of isoforms with at least four different isoforms. Isoforms that were similar to isoforms with a high IgE-reactivity (Bet v 1a = PR-10.01A01) were abundant in all species except B. lenta, while the hypoallergenic isoform Bet v 1d (= PR-10.01B01) was only found in B. pendula and its closest relatives.
Q-TOF LC-MSE allows efficient screening of Bet v 1 isoforms by determining the presence and relative abundance of these isoforms in pollen. B. pendula contains a Bet v 1-mixture in which isoforms with a high and low IgE-reactivity are both abundant. With the possible exception of B. lenta, isoforms identical or very similar to those with a high IgE-reactivity were found in the pollen proteome of all examined birch species. Consequently, these species are also predicted to be allergenic with regard to Bet v 1 related allergies.
Pollen grains induce allergies in susceptible individuals by release of allergens upon contact with mucosal membranes of the upper respiratory tract. We recently demonstrated that pollen not only function as allergen carriers but also as rich sources of bioactive lipids that attract cells involved in allergic inflammation such as neutrophils and eosinophils. Here we demonstrate that soluble factors from birch (Betula alba L.) pollen activate human dendritic cells (DCs) as documented by phenotypical and functional maturation and altered cytokine production. Betula alba L. aqueous pollen extracts (Bet.-APE) selectively inhibited interleukin (IL)-12 p70 production of lipopolysaccharide (LPS)- or CD40L-activated DC, whereas IL-6, IL-10, and TNFα remained unchanged. Presence of Bet.-APE during DC activation resulted in DC with increased T helper type 2 (Th2) cell and reduced Th1 cell polarizing capacity. Chemical analysis of Bet.-APE revealed the presence of phytoprostanes (dinor isoprostanes) with prostaglandin E1-, F1-, A1-, or B1-ring systems of which only E1-phytoprostanes dose dependently inhibited the LPS-induced IL-12 p70 release and augmented the Th2 cell polarizing capacity of DC. These results suggest that pollen-derived E1-phytoprostanes not only resemble endogenous prostaglandin E2 structurally but also functionally in that they act as regulators that modulate human DC function in a fashion that favors Th2 cell polarization.
We describe the role of the BioMagResBank (BMRB) within the Worldwide Protein Data Bank (wwPDB) and recent policies affecting the deposition of biomolecular NMR data. All PDB depositions of structures based on NMR data must now be accompanied by experimental restraints. A scheme has been devised that allows depositors to specify a representative structure and to define residues within that structure found experimentally to be largely unstructured. The BMRB now accepts coordinate sets representing three-dimensional structural models based on experimental NMR data of molecules of biological interest that fall outside the guidelines of the Protein Data Bank (i.e., the molecule is a peptide with 23 or fewer residues, a polynucleotide with 3 or fewer residues, a polysaccharide with 3 or fewer sugar residues, or a natural product), provided that the coordinates are accompanied by representation of the covalent structure of the molecule (atom connectivity), assigned NMR chemical shifts, and the structural restraints used in generating model. The BMRB now contains an archive of NMR data for metabolites and other small molecules found in biological systems.
Archived NMR data; Metabolomics; NMR structure; Structural restraints; Unstructured regions
Local structural similarity restraints (LSSR) provide a novel method for exploiting NCS or structural similarity to an external target structure. Two examples are given where BUSTER re-refinement of PDB entries with LSSR produces marked improvements, enabling further structural features to be modelled.
Maximum-likelihood X-ray macromolecular structure refinement in BUSTER has been extended with restraints facilitating the exploitation of structural similarity. The similarity can be between two or more chains within the structure being refined, thus favouring NCS, or to a distinct ‘target’ structure that remains fixed during refinement. The local structural similarity restraints (LSSR) approach considers all distances less than 5.5 Å between pairs of atoms in the chain to be restrained. For each, the difference from the distance between the corresponding atoms in the related chain is found. LSSR applies a restraint penalty on each difference. A functional form that reaches a plateau for large differences is used to avoid the restraints distorting parts of the structure that are not similar. Because LSSR are local, there is no need to separate out domains. Some restraint pruning is still necessary, but this has been automated. LSSR have been available to academic users of BUSTER since 2009 with the easy-to-use -autoncs and -target target.pdb options. The use of LSSR is illustrated in the re-refinement of PDB entries 5rnt, where -target enables the correct ligand-binding structure to be found, and 1osg, where -autoncs contributes to the location of an additional copy of the cyclic peptide ligand.
BUSTER; NCS restraints; target-structure restraints; local structural similarity restraints
Some future challenges for the PDB and its guardians are discussed and current and future activities in structural bioinformatics at the Protein Data Bank in Europe (PDBe) are described.
The Protein Data Bank in Europe (PDBe) is the European partner in the Worldwide PDB and as such handles depositions of X-ray, NMR and EM data and structure models. PDBe also provides advanced bioinformatics services based on data from the PDB and related resources. Some of the challenges facing the PDB and its guardians are discussed, as well as some of the areas on which PDBe activities will focus in the future (advanced services, ligands, integration, validation and experimental data). Finally, some recent developments at PDBe are described.
Protein Data Bank in Europe