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The Protein Data Bank in Europe (PDBe), a founding member of the Worldwide Protein Data Bank (wwPDB), actively participates in the deposition, curation, validation, archiving and dissemination of macromolecular structure data. PDBe supports diverse research communities in their use of macromolecular structures by enriching the PDB data and by providing advanced tools and services for effective data access, visualization and analysis. This paper details the enrichment of data at PDBe, including mapping of RNA structures to Rfam, and identification of molecules that act as cofactors. PDBe has developed an advanced search facility with â¼100 data categories and sequence searches. New features have been included in the LiteMol viewer at PDBe, with updated visualization of carbohydrates and nucleic acids. Small molecules are now mapped more extensively to external databases and their visual representation has been enhanced. These advances help users to more easily find and interpret macromolecular structure data in order to solve scientific problems.
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Bases de Datos de Proteínas , Programas Informáticos , Análisis por Conglomerados , Exactitud de los Datos , Europa (Continente) , Conformación Proteica , Interfaz Usuario-ComputadorRESUMEN
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
A typical diffraction experiment will generate many images and data sets from different crystals in a very short time. This creates a challenge for the high-throughput operation of modern synchrotron beamlines as well as for the subsequent data processing. Novice users in particular may feel overwhelmed by the tables, plots and numbers that the different data-processing programs and software packages present to them. Here, some of the more common problems that a user has to deal with when processing a set of images that will finally make up a processed data set are shown, concentrating on difficulties that may often show up during the first steps along the path of turning the experiment (i.e. data collection) into a model (i.e. interpreted electron density). Difficulties such as unexpected crystal forms, issues in crystal handling and suboptimal choices of data-collection strategies can often be dealt with, or at least diagnosed, by analysing specific data characteristics during processing. In the end, one wants to distinguish problems over which one has no immediate control once the experiment is finished from problems that can be remedied a posteriori. A new software package, autoPROC, is also presented that combines third-party processing programs with new tools and an automated workflow script that is intended to provide users with both guidance and insight into the offline processing of data affected by the difficulties mentioned above, with particular emphasis on the automated treatment of multi-sweep data sets collected on multi-axis goniostats.
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Automatización de Laboratorios/métodos , Cristalografía por Rayos X/métodos , Programas Informáticos , Procesamiento de Imagen Asistido por ComputadorRESUMEN
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
Aquaporins are required by cells to enable fast adaptation to volume and osmotic changes, as well as microenvironmental metabolic stimuli. Aquaglyceroporins play a crucial role in supplying cancer cells with glycerol for metabolic needs. Here, we show that AQP3 is differentially expressed in cells of a prostate cancer panel. AQP3 is located at the cell membrane and cytoplasm of LNCaP cell while being exclusively expressed in the cytoplasm of Du145 and PC3 cells. LNCaP cells show enhanced hypoxia growth; Du145 and PC3 cells display stress factors, indicating a crucial role for AQP3 at the plasma membrane in adaptation to hypoxia. Hypoxia, both acute and chronic affected AQP3's cellular localization. These outcomes were validated using a machine learning classification approach of the three cell lines and of the six normoxic or hypoxic conditions. Classifiers trained on morphological features derived from cytoskeletal and nuclear labeling alongside corresponding texture features could uniquely identify each individual cell line and the corresponding hypoxia exposure. Cytoskeletal features were 70-90% accurate, while nuclear features allowed for 55-70% accuracy. Cellular texture features (73.9% accuracy) were a stronger predictor of the hypoxic load than the AQP3 distribution (60.3%).
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Acuaporina 3/genética , Neoplasias de la Próstata/genética , Acuaporina 3/metabolismo , Ciclo Celular/genética , Hipoxia de la Célula/genética , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Aprendizaje Automático , Masculino , Neoplasias de la Próstata/patología , Transporte de Proteínas , ARN Mensajero/genética , ARN Mensajero/metabolismoRESUMEN
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
Crystallographic studies of ligands bound to biological macromolecules (proteins and nucleic acids) represent an important source of information concerning drug-target interactions, providing atomic level insights into the physical chemistry of complex formation between macromolecules and ligands. Of the more than 115,000 entries extant in the Protein Data Bank (PDB) archive, â¼75% include at least one non-polymeric ligand. Ligand geometrical and stereochemical quality, the suitability of ligand models for in silico drug discovery and design, and the goodness-of-fit of ligand models to electron-density maps vary widely across the archive. We describe the proceedings and conclusions from the first Worldwide PDB/Cambridge Crystallographic Data Center/Drug Design Data Resource (wwPDB/CCDC/D3R) Ligand Validation Workshop held at the Research Collaboratory for Structural Bioinformatics at Rutgers University on July 30-31, 2015. Experts in protein crystallography from academe and industry came together with non-profit and for-profit software providers for crystallography and with experts in computational chemistry and data archiving to discuss and make recommendations on best practices, as framed by a series of questions central to structural studies of macromolecule-ligand complexes. What data concerning bound ligands should be archived in the PDB? How should the ligands be best represented? How should structural models of macromolecule-ligand complexes be validated? What supplementary information should accompany publications of structural studies of biological macromolecules? Consensus recommendations on best practices developed in response to each of these questions are provided, together with some details regarding implementation. Important issues addressed but not resolved at the workshop are also enumerated.
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Bases de Datos de Proteínas , Proteínas/química , Cristalografía por Rayos X , Curaduría de Datos , Guías como Asunto , Ligandos , Modelos Moleculares , Conformación ProteicaRESUMEN
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.