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The Protein Data Bank in Europe (PDBe, pdbe.org) is actively engaged in the deposition, annotation, remediation, enrichment and dissemination of macromolecular structure data. This paper describes new developments and improvements at PDBe addressing three challenging areas: data enrichment, data dissemination and functional reusability. New features of the PDBe Web site are discussed, including a context dependent menu providing links to raw experimental data and improved presentation of structures solved by hybrid methods. The paper also summarizes the features of the LiteMol suite, which is a set of services enabling fast and interactive 3D visualization of structures, with associated experimental maps, annotations and quality assessment information. We introduce a library of Web components which can be easily reused to port data and functionality available at PDBe to other services. We also introduce updates to the SIFTS resource which maps PDB data to other bioinformatics resources, and the PDBe REST API.
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Biología Computacional/métodos , Bases de Datos de Proteínas , Proteínas/química , Análisis de Secuencia de Proteína/métodos , Interfaz Usuario-Computador , Secuencia de Aminoácidos , Gráficos por Computador , Bases de Datos como Asunto , Europa (Continente) , Humanos , Difusión de la Información , Internet , Modelos Moleculares , Anotación de Secuencia Molecular , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas/genética , Proteínas/metabolismoRESUMEN
The Protein Data Bank in Europe (http://pdbe.org) accepts and annotates depositions of macromolecular structure data in the PDB and EMDB archives and enriches, integrates and disseminates structural information in a variety of ways. The PDBe website has been redesigned based on an analysis of user requirements, and now offers intuitive access to improved and value-added macromolecular structure information. Unique value-added information includes lists of reviews and research articles that cite or mention PDB entries as well as access to figures and legends from full-text open-access publications that describe PDB entries. A powerful new query system not only shows all the PDB entries that match a given query, but also shows the 'best structures' for a given macromolecule, ligand complex or sequence family using data-quality information from the wwPDB validation reports. A PDBe RESTful API has been developed to provide unified access to macromolecular structure data available in the PDB and EMDB archives as well as value-added annotations, e.g. regarding structure quality and up-to-date cross-reference information from the SIFTS resource. Taken together, these new developments facilitate unified access to macromolecular structure data in an intuitive way for non-expert users and support expert users in analysing macromolecular structure data.
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Bases de Datos de Proteínas , Conformación Proteica , Internet , Microscopía Electrónica , Modelos Moleculares , Interfaz Usuario-ComputadorRESUMEN
The synthesis of topologically complex structures, such as links and knots, is one of the current challenges in supramolecular chemistry. The so-called Solomon link consists of two doubly interlocked rings. Despite being a rather simple link from a topological point of view, only few molecular versions of this link have been described so far. Here, we report the quantitative synthesis of a giant molecular Solomon link from 30 subcomponents. The highly charged structure is formed by assembly of 12 cis-blocked Pt(2+) complexes, six Cu(+) ions, and 12 rigid N-donor ligands. Each of the two interlocked rings is composed of six repeating Pt(ligand) units, while the six Cu(+) ions connect the two rings. With a molecular weight of nearly 12â kDa and a diameter of 44.2â Å, this complex is the largest non-DNA-based Solomon link described so far. Furthermore, it represents a molecular version of a "stick link".
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While the Protein Data Bank (PDB) contains a wealth of structural information on ligands bound to macromolecules, their analysis can be challenging due to the large amount and diversity of data. Here, we present PDBe CCDUtils, a versatile toolkit for processing and analysing small molecules from the PDB in PDBx/mmCIF format. PDBe CCDUtils provides streamlined access to all the metadata for small molecules in the PDB and offers a set of convenient methods to compute various properties using RDKit, such as 2D depictions, 3D conformers, physicochemical properties, scaffolds, common fragments, and cross-references to small molecule databases using UniChem. The toolkit also provides methods for identifying all the covalently attached chemical components in a macromolecular structure and calculating similarity among small molecules. By providing a broad range of functionality, PDBe CCDUtils caters to the needs of researchers in cheminformatics, structural biology, bioinformatics and computational chemistry.
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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 5.5â 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.
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Automatización de Laboratorios/métodos , Cristalografía por Rayos X/métodos , Hemoglobinas/análisis , Hemoglobinas/química , Ligandos , Modelos Moleculares , Conformación Molecular , Péptidos Cíclicos/análisis , Péptidos Cíclicos/químicaRESUMEN
Since the outbreak of severe acute respiratory syndrome (SARS) in 2003, the three-dimensional structures of several of the replicase/transcriptase components of SARS coronavirus (SARS-CoV), the non-structural proteins (Nsps), have been determined. However, within the large Nsp3 (1922 amino-acid residues), the structure and function of the so-called SARS-unique domain (SUD) have remained elusive. SUD occurs only in SARS-CoV and the highly related viruses found in certain bats, but is absent from all other coronaviruses. Therefore, it has been speculated that it may be involved in the extreme pathogenicity of SARS-CoV, compared to other coronaviruses, most of which cause only mild infections in humans. In order to help elucidate the function of the SUD, we have determined crystal structures of fragment 389-652 ("SUD(core)") of Nsp3, which comprises 264 of the 338 residues of the domain. Both the monoclinic and triclinic crystal forms (2.2 and 2.8 A resolution, respectively) revealed that SUD(core) forms a homodimer. Each monomer consists of two subdomains, SUD-N and SUD-M, with a macrodomain fold similar to the SARS-CoV X-domain. However, in contrast to the latter, SUD fails to bind ADP-ribose, as determined by zone-interference gel electrophoresis. Instead, the entire SUD(core) as well as its individual subdomains interact with oligonucleotides known to form G-quadruplexes. This includes oligodeoxy- as well as oligoribonucleotides. Mutations of selected lysine residues on the surface of the SUD-N subdomain lead to reduction of G-quadruplex binding, whereas mutations in the SUD-M subdomain abolish it. As there is no evidence for Nsp3 entering the nucleus of the host cell, the SARS-CoV genomic RNA or host-cell mRNA containing long G-stretches may be targets of SUD. The SARS-CoV genome is devoid of G-stretches longer than 5-6 nucleotides, but more extended G-stretches are found in the 3'-nontranslated regions of mRNAs coding for certain host-cell proteins involved in apoptosis or signal transduction, and have been shown to bind to SUD in vitro. Therefore, SUD may be involved in controlling the host cell's response to the viral infection. Possible interference with poly(ADP-ribose) polymerase-like domains is also discussed.
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G-Cuádruplex , ARN Polimerasa Dependiente del ARN/química , ARN Polimerasa Dependiente del ARN/metabolismo , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/química , Proteínas no Estructurales Virales/química , Proteínas no Estructurales Virales/metabolismo , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Cristalografía por Rayos X , Electroforesis , Genoma Viral , Lisina/metabolismo , Datos de Secuencia Molecular , Mutación , Unión Proteica , Conformación Proteica , Pliegue de Proteína , Multimerización de Proteína , Estructura Terciaria de Proteína , ARN Polimerasa Dependiente del ARN/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/genética , Coronavirus Relacionado al Síndrome Respiratorio Agudo Severo/patogenicidad , Proteínas no Estructurales Virales/genética , Replicación ViralRESUMEN
The Worldwide Protein Data Bank (wwPDB) has provided validation reports based on recommendations from community Validation Task Forces for structures in the PDB since 2013. To further enhance validation of small molecules as recommended from the 2016 Ligand Validation Workshop, wwPDB, Global Phasing Ltd., and the Noguchi Institute, recently formed a public/private partnership to incorporate some of their software tools into the wwPDB validation package. Augmented wwPDB validation report features include: two-dimensional (2D) diagrams of small-molecule ligands and carbohydrates, highlighting geometric validation outcomes; 2D topological diagrams of oligosaccharides present in branched entities generated using 2D Symbol Nomenclature for Glycan representation; and views of 3D electron density maps for ligands and carbohydrates, illustrating the goodness-of-fit between the atomic structure and experimental data (X-ray crystallographic structures only). These improvements will impact confidence in ligand conformation and ligand-macromolecular interactions that will aid in understanding biochemical function and contribute to small-molecule drug discovery.
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Carbohidratos/química , Bases de Datos de Proteínas/normas , Simulación del Acoplamiento Molecular/métodos , Proteómica/métodos , Bibliotecas de Moléculas Pequeñas/química , Quimioinformática/métodos , Bases de Datos de Compuestos Químicos/normas , Humanos , Ligandos , Unión Proteica , Proteoma/química , Proteoma/metabolismoRESUMEN
Realising the importance of assessing the quality of the biomolecular structures deposited in the Protein Data Bank (PDB), the Worldwide Protein Data Bank (wwPDB) partners established Validation Task Forces to obtain advice on the methods and standards to be used to validate structures determined by X-ray crystallography, nuclear magnetic resonance spectroscopy and three-dimensional electron cryo-microscopy. The resulting wwPDB validation pipeline is an integral part of the wwPDB OneDep deposition, biocuration and validation system. The wwPDB Validation Service webserver (https://validate.wwpdb.org) can be used to perform checks prior to deposition. Here, it is shown how validation metrics can be combined to produce an overall score that allows the ranking of macromolecular structures and domains in search results. The ValTrendsDB database provides users with a convenient way to access and analyse validation information and other properties of X-ray crystal structures in the PDB, including investigating trends in and correlations between different structure properties and validation metrics.
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Bases de Datos de Proteínas/normas , Internet , Sustancias Macromoleculares/química , Conformación Proteica , Proteínas/análisis , Interfaz Usuario-Computador , Microscopía por Crioelectrón , Curaduría de Datos , Humanos , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Proteínas/químicaRESUMEN
Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) play a crucial role in structure-guided drug discovery and design, and also provide atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. The quality with which small-molecule ligands have been modelled in Protein Data Bank (PDB) entries has been, and continues to be, a matter of concern for many investigators. Correctly interpreting whether electron density found in a binding site is compatible with the soaked or co-crystallized ligand or represents water or buffer molecules is often far from trivial. The Worldwide PDB validation report (VR) provides a mechanism to highlight any major issues concerning the quality of the data and the model at the time of deposition and annotation, so the depositors can fix issues, resulting in improved data quality. The ligand-validation methods used in the generation of the current VRs are described in detail, including an examination of the metrics to assess both geometry and electron-density fit. It is found that the LLDF score currently used to identify ligand electron-density fit outliers can give misleading results and that better ligand-validation metrics are required.
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Bases de Datos de Proteínas , Sustancias Macromoleculares/química , Conformación Proteica , Proteínas/análisis , Proteínas/química , Cristalografía por Rayos X , Humanos , Ligandos , Modelos Moleculares , Estructura MolecularRESUMEN
Database URL: https://www.wwpdb.org/.
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Curaduría de Datos , Bases de Datos de Proteínas , Conformación Proteica , Vocabulario ControladoRESUMEN
The Worldwide PDB recently launched a deposition, biocuration, and validation tool: OneDep. At various stages of OneDep data processing, validation reports for three-dimensional structures of biological macromolecules are produced. These reports are based on recommendations of expert task forces representing crystallography, nuclear magnetic resonance, and cryoelectron microscopy communities. The reports provide useful metrics with which depositors can evaluate the quality of the experimental data, the structural model, and the fit between them. The validation module is also available as a stand-alone web server and as a programmatically accessible web service. A growing number of journals require the official wwPDB validation reports (produced at biocuration) to accompany manuscripts describing macromolecular structures. Upon public release of the structure, the validation report becomes part of the public PDB archive. Geometric quality scores for proteins in the PDB archive have improved over the past decade.
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Bases de Datos de Proteínas/normas , Estudios de Validación como Asunto , Análisis de Secuencia de Proteína/métodos , Análisis de Secuencia de Proteína/normasRESUMEN
Reversible control of the conformation of proteins was employed to probe the relationship between flexibility and specificity of the basic helix-loop-helix protein MyoD. A fusion protein (apaMyoD) was designed where the basic DNA binding helix of MyoD was stablized by an amino-terminal extension with a sequence derived from the bee venom peptide apamin. The disulfide-stabilized helix from apamin served as a nucleus for a helix that extended for a further ten residues, thereby holding apaMyoD's DNA recognition helix in a predominantly alpha-helical conformation. The thermal stability of the DNA complexes of apaMyoD was increased by 13 degrees C relative to MyoD-bHLH. Measurements of the fluorescence anisotropy change on DNA binding indicated that apaMyoD bound to E-box-containing DNA sequences with enhanced affinity relative to MyoD-bHLH. Consequently, the DNA binding specificity of apaMyoD was increased 10-fold.
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Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Anisotropía , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Sitios de Unión , Dicroismo Circular , ADN/genética , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Espectroscopía de Resonancia Magnética , Microscopía Fluorescente/instrumentación , Modelos Moleculares , Datos de Secuencia Molecular , Unión Proteica , Relación Estructura-Actividad , Especificidad por Sustrato , Temperatura , Factores de Transcripción/química , Factores de Transcripción/genéticaRESUMEN
Reversible photocontrol of peptide and protein conformation could prove to be a powerful tool for probing function in diverse biological systems. Here, we report reversible photoswitching of the helix content in short peptides containing an azobenzene cross-linker between cysteine residues at positions i, i + 4, or i, i + 11 in the sequence. Trans-to-cis photoisomerization significantly increases the helix content in the i, i + 4 case and significantly decreases the helix content in the i, i + 11 case. These cross-linker designs significantly expand the possibilities for photocontrol of peptide and protein structure.
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Compuestos Azo/química , Reactivos de Enlaces Cruzados/química , Péptidos/química , Modelos Moleculares , Estructura Secundaria de Proteína , EstereoisomerismoAsunto(s)
Proteínas de Unión al ADN/química , ADN/química , Proteína MioD/química , Secuencia de Aminoácidos , Compuestos Azo/química , Dicroismo Circular , ADN/metabolismo , Proteínas de Unión al ADN/metabolismo , Modelos Moleculares , Datos de Secuencia Molecular , Proteína MioD/metabolismo , Fotoquímica , EspectrofotometríaRESUMEN
Structural and kinetic properties of the human 2-enoyl thioester reductase [mitochondrial enoyl-coenzyme A reductase (MECR)/ETR1] of the mitochondrial fatty acid synthesis (FAS) II pathway have been determined. The crystal structure of this dimeric enzyme (at 2.4 A resolution) suggests that the binding site for the recognition helix of the acyl carrier protein is in a groove between the two adjacent monomers. This groove is connected via the pantetheine binding cleft to the active site. The modeled mode of NADPH binding, using molecular dynamics calculations, suggests that Tyr94 and Trp311 are critical for catalysis, which is supported by enzyme kinetic data. A deep, water-filled pocket, shaped by hydrophobic and polar residues and extending away from the catalytic site, was recognized. This pocket can accommodate a fatty acyl tail of up to 16 carbons. Mutagenesis of the residues near the end of this pocket confirms the importance of this region for the binding of substrate molecules with long fatty acyl tails. Furthermore, the kinetic analysis of the wild-type MECR/ETR1 shows a bimodal distribution of catalytic efficiencies, in agreement with the notion that two major products are generated by the mitochondrial FAS II pathway.
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NADH NADPH Oxidorreductasas/química , Proteína Transportadora de Acilo/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Dominio Catalítico , Cristalografía por Rayos X , Dimerización , Acido Graso Sintasa Tipo I/química , Acido Graso Sintasa Tipo I/genética , Acido Graso Sintasa Tipo I/metabolismo , Humanos , Cinética , Mitocondrias/enzimología , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , NADH NADPH Oxidorreductasas/genética , NADH NADPH Oxidorreductasas/metabolismo , NADP/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH , Conformación Proteica , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología de Secuencia de Aminoácido , Especificidad por SustratoRESUMEN
Synthetic photocontrolled proteins could be powerful tools for probing cellular chemistry. Several previous attempts to produce such systems by incorporating photoisomerizable chromophores into biomolecules have led to photocontrol but with incomplete reversibility, where the chromophore becomes trapped in one photoisomeric state. We report here the design of a modified GCN4-bZIP DNA-binding protein with an azobenzene chromophore introduced between Cys residues at positions 262 and 269 (S262C, N269C) within the zipper domain. As predicted, the trans form of the chromophore destabilizes the helical structure of the coiled-coil region of GCN4-bZIP, leading to diminished DNA binding relative to wild type. Trans-to-cis photoisomerization of the chromophore increases helical content and substantially enhances DNA binding. The system is observed to be readily reversible; thermal relaxation of the chromophore to the trans state and concomitant dissociation of the protein-DNA complex occurs with tau(1/2) approximately 10 min at 37 degrees C. It appears that conformational dynamics in the zipper domain make the transition state for isomerization readily available so that retention of reversible switching is observed.
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Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/metabolismo , Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Aminoácidos , Secuencia de Bases , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/química , Dicroismo Circular , Cartilla de ADN , Proteínas de Unión al ADN/química , Electroforesis Capilar , Cinética , Modelos Moleculares , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Fotoquímica , Unión Proteica , Proteínas de Saccharomyces cerevisiae/química , Factores de Transcripción/químicaRESUMEN
Control of DNA binding of HDH-3, a 18-residue polypeptide based on the recognition helix of the Q50K engrailed homeodomain, has been achieved. HDH-3 was linked to an azobenzene cross-linker through two cysteine residues in an i, i + 11 spacing. For the thermodynamically stable trans configuration of the cross-linker, the dark-adapted peptide (dad-HDH-3) adopted a mainly alpha-helical structure as judged by circular dichroism (CD) spectroscopy. After irradiation with light of 360 nm, the helical content of the peptide (irrad-HDH-3) was reduced significantly and the CD spectrum of the irradiated peptide resembled that of the largely unstructured, unalkylated peptide. Despite lacking helices-1 and -2 and the N-terminal arm of Q50K engrailed, dad-HDH-3 bound to its natural DNA target sequence TAATCC (QRE) with high affinity (K(D) = 7.5 +/- 1.3 nM). The binding affinity for the mutant DNA sequence, TAATTA (ERE), was reduced significantly (K(D) = 140 +/- 11 nM). Unlike irrad-HDH-3, which like the unalkylated parent peptide displayed only marginal DNA binding specificity, dad-HDH-3 specified base pairs 5 and 6 of QRE with an accuracy rivaling that of the intact wild-type Q50K engrailed homeodomain, making dad-HDH-3 the most specific designed DNA binding miniature homeodomain reported to date. Moreover, DNA binding affinity and specificity of HDH-3 could be controlled externally by irradiation with light.
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ADN/química , Proteínas de Homeodominio/química , Péptidos/química , Compuestos Azo , Dicroismo Circular , Reactivos de Enlaces Cruzados , ADN/metabolismo , Proteínas de Unión al ADN/química , Polarización de Fluorescencia , FotoquímicaRESUMEN
A peptide NTH-18 was synthesized in which a N-terminal helix is stabilised by two crossed disulfide bonds to a C-terminal extension. The design was inspired by the structure of the neurotoxic peptide apamin, which has previously been used to stabilise helices in miniature enzymes. CD- and NMR-spectroscopy indicated that NTH-18 adopted a fold similar to that found in apamin. However, the arrangement of the elements of secondary structures was inverted relative to apamin; a N-terminal alpha-helix was connected by a reverse turn to a C-terminal extension of non-canonical secondary structure. NTH-18 displayed significant stability to heat and changes of pH. The high definition of the N-terminal end of the alpha-helix of NTH-18 should make this peptide a useful vehicle to stabilise alpha-helices in proteins with applications in protein engineering and molecular recognition.
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Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Cromatografía Líquida de Alta Presión , Dicroismo Circular , Modelos Moleculares , Datos de Secuencia Molecular , Resonancia Magnética Nuclear Biomolecular , Fragmentos de Péptidos/síntesis química , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
We have designed and synthesized a water-soluble, sulfonated version of an azobenzene-based thiol-reactive cross-linker that can be introduced into peptides and proteins and act as a conformational photoswitch. The sulfonated compound is shown to effect a similar degree of conformational control on a model peptide helix system, as its nonsulfonated counterpart but can be introduced without the need for any organic cosolvent. The sulfonated azobenzene cross-linker thus expands the range of proteins to which photocontrol can be applied.
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Compuestos Azo/química , Reactivos de Enlaces Cruzados/química , Péptidos/química , Agua/química , Secuencia de Aminoácidos , Aminoácidos/química , Compuestos Azo/síntesis química , Dicroismo Circular , Modelos Químicos , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Fotoquímica , Conformación Proteica , Espectrofotometría Ultravioleta , Estereoisomerismo , TermodinámicaRESUMEN
Photo-control of protein conformation could prove useful for probing function in diverse biological systems. Recently, we reported photo-switching of helix content in a short peptide containing an azobenzene cross-linker between cysteine residues at positions i and i + 7 in the sequence. In the original sequence, underlying residues at positions i + 3 and i + 4 were made bulky as preliminary modelling suggested that this would enhance photo-control of helix content. To test this hypothesis, peptides with Val, Aib; Ile, Aib; and Ala, Ala at positions i + 3 and i + 4 were synthesized, cross-linked and characterized. Before cross-linking, the peptides show distinct conformational behaviours: two with differing helix/coil mixtures whereas the other has a circular dichroism (CD) spectrum characteristic of beta-sheet and a tendency to aggregate. However, upon cross-linking the peptides have very similar CD spectra: predominantly random coil in the dark but predominantly helical upon irradiation. These results refute the original hypothesis. Steric interactions between the linker and underlying residues do not appear to be critical for photo-switching behaviour. When the cross-linking bridge is lengthened by replacing the i, i + 7 cysteine residues with homocysteine, a lower degree of photo-control of helicity is observed. Furthermore, a non-cross-linking version of the azobenzene reagent is shown not to produce any photo-control of helicity. We conclude that the intramolecular cross-link is essential for photo-switching and that it should be applicable to a wide range of peptides and proteins.