PDBe: improved findability of macromolecular structure data in the PDB.

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 ∼100 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.

PDBe: towards reusable data delivery infrastructure at protein data bank in Europe.

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.

Crystal structure of the entire respiratory complex I.

Complex I is the first and largest enzyme of the respiratory chain and has a central role in cellular energy production through the coupling of NADH:ubiquinone electron transfer to proton translocation. It is also implicated in many common human neurodegenerative diseases. Here, we report the first crystal structure of the entire, intact complex I (from Thermus thermophilus) at 3.3 Å resolution. The structure of the 536-kDa complex comprises 16 different subunits, with a total of 64 transmembrane helices and 9 iron-sulphur clusters. The core fold of subunit Nqo8 (ND1 in humans) is, unexpectedly, similar to a half-channel of the antiporter-like subunits. Small subunits nearby form a linked second half-channel, which completes the fourth proton-translocation pathway (present in addition to the channels in three antiporter-like subunits). The quinone-binding site is unusually long, narrow and enclosed. The quinone headgroup binds at the deep end of this chamber, near iron-sulphur cluster N2. Notably, the chamber is linked to the fourth channel by a 'funnel' of charged residues. The link continues over the entire membrane domain as a flexible central axis of charged and polar residues, and probably has a leading role in the propagation of conformational changes, aided by coupling elements. The structure suggests that a unique, out-of-the-membrane quinone-reaction chamber enables the redox energy to drive concerted long-range conformational changes in the four antiporter-like domains, resulting in translocation of four protons per cycle.

PDBe: improved accessibility of macromolecular structure data from PDB and EMDB.

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.

Structure of bacterial respiratory complex I.

Complex I (NADH:ubiquinone oxidoreductase) plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. It is the largest protein assembly of respiratory chains and one of the most elaborate redox membrane proteins known. Bacterial enzyme is about half the size of mitochondrial and thus provides its important "minimal" model. Dysfunction of mitochondrial complex I is implicated in many human neurodegenerative diseases. The L-shaped complex consists of a hydrophilic arm, where electron transfer occurs, and a membrane arm, where proton translocation takes place. We have solved the crystal structures of the hydrophilic domain of complex I from Thermus thermophilus, the membrane domain from Escherichia coli and recently of the intact, entire complex I from T. thermophilus (536 kDa, 16 subunits, 9 iron-sulphur clusters, 64 transmembrane helices). The 95Å long electron transfer pathway through the enzyme proceeds from the primary electron acceptor flavin mononucleotide through seven conserved Fe-S clusters to the unusual elongated quinone-binding site at the interface with the membrane domain. Four putative proton translocation channels are found in the membrane domain, all linked by the central flexible axis containing charged residues. The redox energy of electron transfer is coupled to proton translocation by the as yet undefined mechanism proposed to involve long-range conformational changes. This article is part of a Special Issue entitled Respiratory complex I, edited by Volker Zickermann and Ulrich Brandt.

PDBe: Protein Data Bank in Europe.

The Protein Data Bank in Europe (pdbe.org) is a founding member of the Worldwide PDB consortium (wwPDB; wwpdb.org) and as such is actively engaged in the deposition, annotation, remediation and dissemination of macromolecular structure data through the single global archive for such data, the PDB. Similarly, PDBe is a member of the EMDataBank organisation (emdatabank.org), which manages the EMDB archive for electron microscopy data. PDBe also develops tools that help the biomedical science community to make effective use of the data in the PDB and EMDB for their research. Here we describe new or improved services, including updated SIFTS mappings to other bioinformatics resources, a new browser for the PDB archive based on Gene Ontology (GO) annotation, updates to the analysis of Nuclear Magnetic Resonance-derived structures, redesigned search and browse interfaces, and new or updated visualisation and validation tools for EMDB entries.

Community recommendations on cryoEM data archiving and validation: Outcomes of a wwPDB/EMDB workshop on cryoEM data management, deposition and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and consensus recommendations resulting from the workshop. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

Community recommendations on cryoEM data archiving and validation.

In January 2020, a workshop was held at EMBL-EBI (Hinxton, UK) to discuss data requirements for the deposition and validation of cryoEM structures, with a focus on single-particle analysis. The meeting was attended by 47 experts in data processing, model building and refinement, validation, and archiving of such structures. This report describes the workshop's motivation and history, the topics discussed, and the resulting consensus recommendations. Some challenges for future methods-development efforts in this area are also highlighted, as is the implementation to date of some of the recommendations.

A long road towards the structure of respiratory complex I, a giant molecular proton pump.

Complex I (NADH:ubiquinone oxidoreductase) is central to cellular energy production, being the first and largest enzyme of the respiratory chain in mitochondria. It couples electron transfer from NADH to ubiquinone with proton translocation across the inner mitochondrial membrane and is involved in a wide range of human neurodegenerative disorders. Mammalian complex I is composed of 44 different subunits, whereas the 'minimal' bacterial version contains 14 highly conserved 'core' subunits. The L-shaped assembly consists of hydrophilic and membrane domains. We have determined all known atomic structures of complex I, starting from the hydrophilic domain of Thermus thermophilus enzyme (eight subunits, nine Fe-S clusters), followed by the membrane domains of the Escherichia coli (six subunits, 55 transmembrane helices) and T. thermophilus (seven subunits, 64 transmembrane helices) enzymes, and finally culminating in a recent crystal structure of the entire intact complex I from T. thermophilus (536 kDa, 16 subunits, nine Fe-S clusters, 64 transmembrane helices). The structure suggests an unusual and unique coupling mechanism via long-range conformational changes. Determination of the structure of the entire complex was possible only through this step-by-step approach, building on from smaller subcomplexes towards the entire assembly. Large membrane proteins are notoriously difficult to crystallize, and so various non-standard and sometimes counterintuitive approaches were employed in order to achieve crystal diffraction to high resolution and solve the structures. These steps, as well as the implications from the final structure, are discussed in the present review.

The CCP4 suite: integrative software for macromolecular crystallography.

The Collaborative Computational Project No. 4 (CCP4) is a UK-led international collective with a mission to develop, test, distribute and promote software for macromolecular crystallography. The CCP4 suite is a multiplatform collection of programs brought together by familiar execution routines, a set of common libraries and graphical interfaces. The CCP4 suite has experienced several considerable changes since its last reference article, involving new infrastructure, original programs and graphical interfaces. This article, which is intended as a general literature citation for the use of the CCP4 software suite in structure determination, will guide the reader through such transformations, offering a general overview of the new features and outlining future developments. As such, it aims to highlight the individual programs that comprise the suite and to provide the latest references to them for perusal by crystallographers around the world.

PDBx/mmCIF Ecosystem: Foundational Semantic Tools for Structural Biology.

PDBx/mmCIF, Protein Data Bank Exchange (PDBx) macromolecular Crystallographic Information Framework (mmCIF), has become the data standard for structural biology. With its early roots in the domain of small-molecule crystallography, PDBx/mmCIF provides an extensible data representation that is used for deposition, archiving, remediation, and public dissemination of experimentally determined three-dimensional (3D) structures of biological macromolecules by the Worldwide Protein Data Bank (wwPDB, wwpdb.org). Extensions of PDBx/mmCIF are similarly used for computed structure models by ModelArchive (modelarchive.org), integrative/hybrid structures by PDB-Dev (pdb-dev.wwpdb.org), small angle scattering data by Small Angle Scattering Biological Data Bank SASBDB (sasbdb.org), and for models computed generated with the AlphaFold 2.0 deep learning software suite (alphafold.ebi.ac.uk). Community-driven development of PDBx/mmCIF spans three decades, involving contributions from researchers, software and methods developers in structural sciences, data repository providers, scientific publishers, and professional societies. Having a semantically rich and extensible data framework for representing a wide range of structural biology experimental and computational results, combined with expertly curated 3D biostructure data sets in public repositories, accelerates the pace of scientific discovery. Herein, we describe the architecture of the PDBx/mmCIF data standard, tools used to maintain representations of the data standard, governance, and processes by which data content standards are extended, plus community tools/software libraries available for processing and checking the integrity of PDBx/mmCIF data. Use cases exemplify how the members of the Worldwide Protein Data Bank have used PDBx/mmCIF as the foundation for its pipeline for delivering Findable, Accessible, Interoperable, and Reusable (FAIR) data to many millions of users worldwide.

Structural basis for the mechanism of respiratory complex I.

Complex I plays a central role in cellular energy production, coupling electron transfer between NADH and quinone to proton translocation. The mechanism of this highly efficient enzyme is currently unknown. Mitochondrial complex I is a major source of reactive oxygen species, which may be one of the causes of aging. Dysfunction of complex I is implicated in many human neurodegenerative diseases. We have determined several x-ray structures of the oxidized and reduced hydrophilic domain of complex I from Thermus thermophilus at up to 3.1 A resolution. The structures reveal the mode of interaction of complex I with NADH, explaining known kinetic data and providing implications for the mechanism of reactive oxygen species production at the flavin site of complex I. Bound metals were identified in the channel at the interface with the frataxin-like subunit Nqo15, indicating possible iron-binding sites. Conformational changes upon reduction of the complex involve adjustments in the nucleotide-binding pocket, as well as small but significant shifts of several alpha-helices at the interface with the membrane domain. These shifts are likely to be driven by the reduction of nearby iron-sulfur clusters N2 and N6a/b. Cluster N2 is the electron donor to quinone and is coordinated by unique motif involving two consecutive (tandem) cysteines. An unprecedented "on/off switch" (disconnection) of coordinating bonds between the tandem cysteines and this cluster was observed upon reduction. Comparison of the structures suggests a novel mechanism of coupling between electron transfer and proton translocation, combining conformational changes and protonation/deprotonation of tandem cysteines.

Chemical and NADH-induced, ROS-dependent, cross-linking between subunits of complex I from Escherichia coli and Thermus thermophilus.

Complex I of respiratory chains transfers electrons from NADH to ubiquinone, coupled to the translocation of protons across the membrane. Two alternative coupling mechanisms are being discussed, redox-driven or conformation-driven. Using "zero-length" cross-linking reagent and isolated hydrophilic domains of complex I from Escherichia coli and Thermus thermophilus, we show that the pattern of cross-links between subunits changes significantly in the presence of NADH. Similar observations were made previously with intact purified E. coli and bovine complex I. This indicates that, upon reduction with NADH, similar conformational changes are likely to occur in the intact enzyme and in the isolated hydrophilic domain (which can be used for crystallographic studies). Within intact E. coli complex I, the cross-link between the hydrophobic subunits NuoA and NuoJ was abolished in the presence of NADH, indicating that conformational changes extend into the membrane domain, possibly as part of a coupling mechanism. Unexpectedly, in the absence of any chemical cross-linker, incubation of complex I with NADH resulted in covalent cross-links between subunits Nqo4 (NuoCD) and Nqo6 (NuoB), as well as between Nqo6 and Nqo9. Their formation depends on the presence of oxygen and so is likely a result of oxidative damage via reactive oxygen species (ROS) induced cross-linking. In addition, ROS- and metal ion-dependent proteolysis of these subunits (as well as Nqo3) is observed. Fe-S cluster N2 is coordinated between subunits Nqo4 and Nqo6 and could be involved in these processes. Our observations suggest that oxidative damage to complex I in vivo may include not only side-chain modifications but also protein cross-linking and degradation.

Crystallization and preliminary X-ray analysis of recombinant Glomerella cingulata cutinase.

Cutinase catalyzes the hydrolysis of water-soluble esters and long-chain triglycerides and belongs to the family of serine hydrolases. The enzyme is thought to represent an evolutionary link between the esterase and lipase families and has potential applications in a wide range of industrial hydrolytic processes, for which an understanding of the molecular basis of its substrate specificity is critical. Glomerella cingulata cutinase has been cloned and the protein has been overexpressed in Escherichia coli, purified and subsequently crystallized in a wide range of different crystal forms in the presence and absence of inhibitors. The best crystals are those of the apo cutinase, which diffract to beyond 1.6 A resolution and belong to space group P4(1)2(1)2 or P4(3)2(1)2. Crystals of cutinase with the inhibitors PETFP or E600 belong to space groups P2(1)2(1)2(1) and P2(1), respectively, and diffract to approximately 2.5 A resolution. All of the crystals are suitable for structural studies, which are currently ongoing.

Worldwide Protein Data Bank biocuration supporting open access to high-quality 3D structural biology data.

Database URL: https://www.wwpdb.org/.

Evidence supporting a cis-enediol-based mechanism for Pyrococcus furiosus phosphoglucose isomerase.

The enzymatic aldose ketose isomerisation of glucose and fructose sugars involves the transfer of a hydrogen between their C1 and C2 carbon atoms and, in principle, can proceed through either a direct hydride shift or via a cis-enediol intermediate. Pyrococcus furiosus phosphoglucose isomerase (PfPGI), an archaeal metalloenzyme, which catalyses the interconversion of glucose 6-phosphate and fructose 6-phosphate, has been suggested to operate via a hydride shift mechanism. In contrast, the structurally distinct PGIs of eukaryotic or bacterial origin are thought to catalyse isomerisation via a cis-enediol intermediate. We have shown by NMR that hydrogen exchange between substrate and solvent occurs during the reaction catalysed by PfPGI eliminating the possibility of a hydride-shift-based mechanism. In addition, kinetic measurements on this enzyme have shown that 5-phospho-d-arabinonohydroxamate, a stable analogue of the putative cis-enediol intermediate, is the most potent inhibitor of the enzyme yet discovered. Furthermore, determination and analysis of crystal structures of PfPGI with bound zinc and the substrate F6P, and with a number of competitive inhibitors, and EPR analysis of the coordination of the metal ion within PfPGI, have suggested that a cis-enediol intermediate-based mechanism is used by PfPGI with Glu97 acting as the catalytic base responsible for isomerisation.

Validation of Structures in the Protein Data Bank.

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.

OneDep: Unified wwPDB System for Deposition, Biocuration, and Validation of Macromolecular Structures in the PDB Archive.

OneDep, a unified system for deposition, biocuration, and validation of experimentally determined structures of biological macromolecules to the PDB archive, has been developed as a global collaboration by the worldwide PDB (wwPDB) partners. This new system was designed to ensure that the wwPDB could meet the evolving archiving requirements of the scientific community over the coming decades. OneDep unifies deposition, biocuration, and validation pipelines across all wwPDB, EMDB, and BMRB deposition sites with improved focus on data quality and completeness in these archives, while supporting growth in the number of depositions and increases in their average size and complexity. In this paper, we describe the design, functional operation, and supporting infrastructure of the OneDep system, and provide initial performance assessments.

The structures of inhibitor complexes of Pyrococcus furiosus phosphoglucose isomerase provide insights into substrate binding and catalysis.

Pyrococcus furiosus phosphoglucose isomerase (PfPGI) is a metal-containing enzyme that catalyses the interconversion of glucose 6-phosphate (G6P) and fructose 6-phosphate (F6P). The recent structure of PfPGI has confirmed the hypothesis that the enzyme belongs to the cupin superfamily and identified the position of the active site. This fold is distinct from the alphabetaalpha sandwich fold commonly seen in phosphoglucose isomerases (PGIs) that are found in bacteria, eukaryotes and some archaea. Whilst the mechanism of the latter family is thought to proceed through a cis-enediol intermediate, analysis of the structure of PfPGI in the presence of inhibitors has led to the suggestion that the mechanism of this enzyme involves the metal-dependent direct transfer of a hydride between C1 and C2 atoms of the substrate. To gain further insight in the reaction mechanism of PfPGI, the structures of the free enzyme and the complexes with the inhibitor, 5-phospho-d-arabinonate (5PAA) in the presence and absence of metal have been determined. Comparison of these structures with those of equivalent complexes of the eukaryotic PGIs reveals similarities at the active site in the disposition of possible catalytic residues. These include the presence of a glutamic acid residue, Glu97 in PfPGI, which occupies the same position relative to the inhibitor as that of the glutamate that is thought to function as the catalytic base in the eukaryal-type PGIs. These similarities suggest that aspects of the catalytic mechanisms of these two structurally unrelated PGIs may be similar and based on an enediol intermediate.

Small molecule annotation for the Protein Data Bank.

The Protein Data Bank (PDB) is the single global repository for three-dimensional structures of biological macromolecules and their complexes, and its more than 100,000 structures contain more than 20,000 distinct ligands or small molecules bound to proteins and nucleic acids. Information about these small molecules and their interactions with proteins and nucleic acids is crucial for our understanding of biochemical processes and vital for structure-based drug design. Small molecules present in a deposited structure may be attached to a polymer or may occur as a separate, non-covalently linked ligand. During curation of a newly deposited structure by wwPDB annotation staff, each molecule is cross-referenced to the PDB Chemical Component Dictionary (CCD). If the molecule is new to the PDB, a dictionary description is created for it. The information about all small molecule components found in the PDB is distributed via the ftp archive as an external reference file. Small molecule annotation in the PDB also includes information about ligand-binding sites and about covalent and other linkages between ligands and macromolecules. During the remediation of the peptide-like antibiotics and inhibitors present in the PDB archive in 2011, it became clear that additional annotation was required for consistent representation of these molecules, which are quite often composed of several sequential subcomponents including modified amino acids and other chemical groups. The connectivity information of the modified amino acids is necessary for correct representation of these biologically interesting molecules. The combined information is made available via a new resource called the Biologically Interesting molecules Reference Dictionary, which is complementary to the CCD and is now routinely used for annotation of peptide-like antibiotics and inhibitors.