Outcomes of the EMDataResource cryo-EM Ligand Modeling Challenge.

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein-nucleic acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: Escherichia coli beta-galactosidase with inhibitor, SARS-CoV-2 virus RNA-dependent RNA polymerase with covalently bound nucleotide analog and SARS-CoV-2 virus ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. The quality of submitted ligand models and surrounding atoms were analyzed by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics and contact scores. A composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

The Nucleic Acid Knowledgebase: a new portal for 3D structural information about nucleic acids.

The Nucleic Acid Knowledgebase (nakb.org) is a new data resource, updated weekly, for experimentally determined 3D structures containing DNA and/or RNA nucleic acid polymers and their biological assemblies. NAKB indexes nucleic acid-containing structures derived from all major structure determination methods (X-ray, NMR and EM), including all held by the Protein Data Bank (PDB). As the planned successor to the Nucleic Acid Database (NDB), NAKB's design preserves all functionality of the NDB and provides novel nucleic acid-centric content, including structural and functional annotations, as well as annotations from and links to external resources. A variety of custom interactive tools have been developed to enable rapid exploration and drill-down of NAKB's content.

RCSB Protein Data Bank (RCSB.org): delivery of experimentally-determined PDB structures alongside one million computed structure models of proteins from artificial intelligence/machine learning.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), founding member of the Worldwide Protein Data Bank (wwPDB), is the US data center for the open-access PDB archive. As wwPDB-designated Archive Keeper, RCSB PDB is also responsible for PDB data security. Annually, RCSB PDB serves >10 000 depositors of three-dimensional (3D) biostructures working on all permanently inhabited continents. RCSB PDB delivers data from its research-focused RCSB.org web portal to many millions of PDB data consumers based in virtually every United Nations-recognized country, territory, etc. This Database Issue contribution describes upgrades to the research-focused RCSB.org web portal that created a one-stop-shop for open access to ∼200 000 experimentally-determined PDB structures of biological macromolecules alongside >1 000 000 incorporated Computed Structure Models (CSMs) predicted using artificial intelligence/machine learning methods. RCSB.org is a 'living data resource.' Every PDB structure and CSM is integrated weekly with related functional annotations from external biodata resources, providing up-to-date information for the entire corpus of 3D biostructure data freely available from RCSB.org with no usage limitations. Within RCSB.org, PDB structures and the CSMs are clearly identified as to their provenance and reliability. Both are fully searchable, and can be analyzed and visualized using the full complement of RCSB.org web portal capabilities.

Cryo-EM model validation recommendations based on outcomes of the 2019 EMDataResource challenge.

This paper describes outcomes of the 2019 Cryo-EM Model Challenge. The goals were to (1) assess the quality of models that can be produced from cryogenic electron microscopy (cryo-EM) maps using current modeling software, (2) evaluate reproducibility of modeling results from different software developers and users and (3) compare performance of current metrics used for model evaluation, particularly Fit-to-Map metrics, with focus on near-atomic resolution. Our findings demonstrate the relatively high accuracy and reproducibility of cryo-EM models derived by 13 participating teams from four benchmark maps, including three forming a resolution series (1.8 to 3.1 Å). The results permit specific recommendations to be made about validating near-atomic cryo-EM structures both in the context of individual experiments and structure data archives such as the Protein Data Bank. We recommend the adoption of multiple scoring parameters to provide full and objective annotation and assessment of the model, reflective of the observed cryo-EM map density.

RCSB Protein Data Bank: powerful new tools for exploring 3D structures of biological macromolecules for basic and applied research and education in fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences.

The Research Collaboratory for Structural Bioinformatics Protein Data Bank (RCSB PDB), the US data center for the global PDB archive and a founding member of the Worldwide Protein Data Bank partnership, serves tens of thousands of data depositors in the Americas and Oceania and makes 3D macromolecular structure data available at no charge and without restrictions to millions of RCSB.org users around the world, including >660 000 educators, students and members of the curious public using PDB101.RCSB.org. PDB data depositors include structural biologists using macromolecular crystallography, nuclear magnetic resonance spectroscopy, 3D electron microscopy and micro-electron diffraction. PDB data consumers accessing our web portals include researchers, educators and students studying fundamental biology, biomedicine, biotechnology, bioengineering and energy sciences. During the past 2 years, the research-focused RCSB PDB web portal (RCSB.org) has undergone a complete redesign, enabling improved searching with full Boolean operator logic and more facile access to PDB data integrated with >40 external biodata resources. New features and resources are described in detail using examples that showcase recently released structures of SARS-CoV-2 proteins and host cell proteins relevant to understanding and addressing the COVID-19 global pandemic.

Evolution of standardization and dissemination of cryo-EM structures and data jointly by the community, PDB, and EMDB.

Cryogenic electron microscopy (cryo-EM) methods began to be used in the mid-1970s to study thin and periodic arrays of proteins. Following a half-century of development in cryo-specimen preparation, instrumentation, data collection, data processing, and modeling software, cryo-EM has become a routine method for solving structures from large biological assemblies to small biomolecules at near to true atomic resolution. This review explores the critical roles played by the Protein Data Bank (PDB) and Electron Microscopy Data Bank (EMDB) in partnership with the community to develop the necessary infrastructure to archive cryo-EM maps and associated models. Public access to cryo-EM structure data has in turn facilitated better understanding of structure-function relationships and advancement of image processing and modeling tool development. The partnership between the global cryo-EM community and PDB and EMDB leadership has synergistically shaped the standards for metadata, one-stop deposition of maps and models, and validation metrics to assess the quality of cryo-EM structures. The advent of cryo-electron tomography (cryo-ET) for in situ molecular cell structures at a broad resolution range and their correlations with other imaging data introduce new data archival challenges in terms of data size and complexity in the years to come.

Anticipating innovations in structural biology.

In this review, we describe how the interplay among science, technology and community interests contributed to the evolution of four structural biology data resources. We present the method by which data deposited by scientists are prepared for worldwide distribution, and argue that data archiving in a trusted repository must be an integral part of any scientific investigation.

The RNA Polymerase α Subunit Recognizes the DNA Shape of the Upstream Promoter Element.

We demonstrate here that the α subunit C-terminal domain of Escherichia coli RNA polymerase (αCTD) recognizes the upstream promoter (UP) DNA element via its characteristic minor groove shape and electrostatic potential. In two compositionally distinct crystallized assemblies, a pair of αCTD subunits bind in tandem to the UP element consensus A-tract that is 6 bp in length (A6-tract), each with their arginine 265 guanidinium group inserted into the minor groove. The A6-tract minor groove is significantly narrowed in these crystal structures, as well as in computationally predicted structures of free and bound DNA duplexes derived by Monte Carlo and molecular dynamics simulations, respectively. The negative electrostatic potential of free A6-tract DNA is substantially enhanced compared to that of generic DNA. Shortening the A-tract by 1 bp is shown to "knock out" binding of the second αCTD through widening of the minor groove. Furthermore, in computationally derived structures with arginine 265 mutated to alanine in either αCTD, either with or without the "knockout" DNA mutation, contact with the DNA is perturbed, highlighting the importance of arginine 265 in achieving αCTD-DNA binding. These results demonstrate that the importance of the DNA shape in sequence-dependent recognition of DNA by RNA polymerase is comparable to that of certain transcription factors.

Evaluation system and web infrastructure for the second cryo-EM model challenge.

An evaluation system and a web infrastructure were developed for the second cryo-EM model challenge. The evaluation system includes tools to validate stereo-chemical plausibility of submitted models, check their fit to the corresponding density maps, estimate their overall and per-residue accuracy, and assess their similarity to reference cryo-EM or X-ray structures as well as other models submitted in this challenge. The web infrastructure provides a convenient interface for analyzing models at different levels of detail. It includes interactively sortable tables of evaluation scores for different subsets of models and different sublevels of structure organization, and a suite of visualization tools facilitating model analysis. The results are publicly accessible at http://model-compare.emdatabank.org.

EMDataBank unified data resource for 3DEM.

Three-dimensional Electron Microscopy (3DEM) has become a key experimental method in structural biology for a broad spectrum of biological specimens from molecules to cells. The EMDataBank project provides a unified portal for deposition, retrieval and analysis of 3DEM density maps, atomic models and associated metadata (emdatabank.org). We provide here an overview of the rapidly growing 3DEM structural data archives, which include maps in EM Data Bank and map-derived models in the Protein Data Bank. In addition, we describe progress and approaches toward development of validation protocols and methods, working with the scientific community, in order to create a validation pipeline for 3DEM data.

IHMCIF: An Extension of the PDBx/mmCIF Data Standard for Integrative Structure Determination Methods.

IHMCIF (github.com/ihmwg/IHMCIF) is a data information framework that supports archiving and disseminating macromolecular structures determined by integrative or hybrid modeling (IHM), and making them Findable, Accessible, Interoperable, and Reusable (FAIR). IHMCIF is an extension of the Protein Data Bank Exchange/macromolecular Crystallographic Information Framework (PDBx/mmCIF) that serves as the framework for the Protein Data Bank (PDB) to archive experimentally determined atomic structures of biological macromolecules and their complexes with one another and small molecule ligands (e.g., enzyme cofactors and drugs). IHMCIF serves as the foundational data standard for the PDB-Dev prototype system, developed for archiving and disseminating integrative structures. It utilizes a flexible data representation to describe integrative structures that span multiple spatiotemporal scales and structural states with definitions for restraints from a variety of experimental methods contributing to integrative structural biology. The IHMCIF extension was created with the benefit of considerable community input and recommendations gathered by the Worldwide Protein Data Bank (wwPDB) Task Force for Integrative or Hybrid Methods (wwpdb.org/task/hybrid). Herein, we describe the development of IHMCIF to support evolving methodologies and ongoing advancements in integrative structural biology. Ultimately, IHMCIF will facilitate the unification of PDB-Dev data and tools with the PDB archive so that integrative structures can be archived and disseminated through PDB.

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.

Outcomes of the EMDataResource Cryo-EM Ligand Modeling Challenge.

The EMDataResource Ligand Model Challenge aimed to assess the reliability and reproducibility of modeling ligands bound to protein and protein/nucleic-acid complexes in cryogenic electron microscopy (cryo-EM) maps determined at near-atomic (1.9-2.5 Å) resolution. Three published maps were selected as targets: E. coli beta-galactosidase with inhibitor, SARS-CoV-2 RNA-dependent RNA polymerase with covalently bound nucleotide analog, and SARS-CoV-2 ion channel ORF3a with bound lipid. Sixty-one models were submitted from 17 independent research groups, each with supporting workflow details. We found that (1) the quality of submitted ligand models and surrounding atoms varied, as judged by visual inspection and quantification of local map quality, model-to-map fit, geometry, energetics, and contact scores, and (2) a composite rather than a single score was needed to assess macromolecule+ligand model quality. These observations lead us to recommend best practices for assessing cryo-EM structures of liganded macromolecules reported at near-atomic resolution.

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.

EMDataBank.org: unified data resource for CryoEM.

Cryo-electron microscopy reconstruction methods are uniquely able to reveal structures of many important macromolecules and macromolecular complexes. EMDataBank.org, a joint effort of the Protein Data Bank in Europe (PDBe), the Research Collaboratory for Structural Bioinformatics (RCSB) and the National Center for Macromolecular Imaging (NCMI), is a global 'one-stop shop' resource for deposition and retrieval of cryoEM maps, models and associated metadata. The resource unifies public access to the two major archives containing EM-based structural data: EM Data Bank (EMDB) and Protein Data Bank (PDB), and facilitates use of EM structural data of macromolecules and macromolecular complexes by the wider scientific community.

Three-dimensional EM structure of an intact activator-dependent transcription initiation complex.

We present the experimentally determined 3D structure of an intact activator-dependent transcription initiation complex comprising the Escherichia coli catabolite activator protein (CAP), RNA polymerase holoenzyme (RNAP), and a DNA fragment containing positions -78 to +20 of a Class I CAP-dependent promoter with a CAP site at position -61.5 and a premelted transcription bubble. A 20-A electron microscopy reconstruction was obtained by iterative projection-based matching of single particles visualized in carbon-sandwich negative stain and was fitted using atomic coordinate sets for CAP, RNAP, and DNA. The structure defines the organization of a Class I CAP-RNAP-promoter complex and supports previously proposed interactions of CAP with RNAP alpha subunit C-terminal domain (alphaCTD), interactions of alphaCTD with sigma(70) region 4, interactions of CAP and RNAP with promoter DNA, and phased-DNA-bend-dependent partial wrapping of DNA around the complex. The structure also reveals the positions and shapes of species-specific domains within the RNAP beta', beta, and sigma(70) subunits.

Developing Community Resources for Nucleic Acid Structures.

In this review, we describe the creation of the Nucleic Acid Database (NDB) at Rutgers University and how it became a testbed for the current infrastructure of the RCSB Protein Data Bank. We describe some of the special features of the NDB and how it has been used to enable research. Plans for the next phase as the Nucleic Acid Knowledgebase (NAKB) are summarized.

Electron microscopy holdings of the Protein Data Bank: the impact of the resolution revolution, new validation tools, and implications for the future.

As a discipline, structural biology has been transformed by the three-dimensional electron microscopy (3DEM) "Resolution Revolution" made possible by convergence of robust cryo-preservation of vitrified biological materials, sample handling systems, and measurement stages operating a liquid nitrogen temperature, improvements in electron optics that preserve phase information at the atomic level, direct electron detectors (DEDs), high-speed computing with graphics processing units, and rapid advances in data acquisition and processing software. 3DEM structure information (atomic coordinates and related metadata) are archived in the open-access Protein Data Bank (PDB), which currently holds more than 11,000 3DEM structures of proteins and nucleic acids, and their complexes with one another and small-molecule ligands (~ 6% of the archive). Underlying experimental data (3DEM density maps and related metadata) are stored in the Electron Microscopy Data Bank (EMDB), which currently holds more than 21,000 3DEM density maps. After describing the history of the PDB and the Worldwide Protein Data Bank (wwPDB) partnership, which jointly manages both the PDB and EMDB archives, this review examines the origins of the resolution revolution and analyzes its impact on structural biology viewed through the lens of PDB holdings. Six areas of focus exemplifying the impact of 3DEM across the biosciences are discussed in detail (icosahedral viruses, ribosomes, integral membrane proteins, SARS-CoV-2 spike proteins, cryogenic electron tomography, and integrative structure determination combining 3DEM with complementary biophysical measurement techniques), followed by a review of 3DEM structure validation by the wwPDB that underscores the importance of community engagement.

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.