Comparing Cryo-EM Reconstructions and Validating Atomic Model Fit Using Difference Maps.

Cryogenic electron microscopy (cryo-EM) is a powerful technique for determining structures of multiple conformational or compositional states of macromolecular assemblies involved in cellular processes. Recent technological developments have led to a leap in the resolution of many cryo-EM data sets, making atomic model building more common for data interpretation. We present a method for calculating differences between two cryo-EM maps or a map and a fitted atomic model. The proposed approach works by scaling the maps using amplitude matching in resolution shells. To account for variability in local resolution of cryo-EM data, we include a procedure for local amplitude scaling that enables appropriate scaling of local map contrast. The approach is implemented as a user-friendly tool in the CCP-EM software package. To obtain clean and interpretable differences, we propose a protocol involving steps to process the input maps and output differences. We demonstrate the utility of the method for identifying conformational and compositional differences including ligands. We also highlight the use of difference maps for evaluating atomic model fit in cryo-EM maps.

Building bridges between cellular and molecular structural biology.

The integration of cellular and molecular structural data is key to understanding the function of macromolecular assemblies and complexes in their in vivo context. Here we report on the outcomes of a workshop that discussed how to integrate structural data from a range of public archives. The workshop identified two main priorities: the development of tools and file formats to support segmentation (that is, the decomposition of a three-dimensional volume into regions that can be associated with defined objects), and the development of tools to support the annotation of biological structures.

Improved metrics for comparing structures of macromolecular assemblies determined by 3D electron-microscopy.

Recent developments in 3-dimensional electron microcopy (3D-EM) techniques and a concomitant drive to look at complex molecular structures, have led to a rapid increase in the amount of volume data available for biomolecules. This creates a demand for better methods to analyse the data, including improved scores for comparison, classification and integration of data at different resolutions. To this end, we developed and evaluated a set of scoring functions that compare 3D-EM volumes. To test our scores we used a benchmark set of volume alignments derived from the Electron Microscopy Data Bank. We find that the performance of different scores vary with the map-type, resolution and the extent of overlap between volumes. Importantly, adding the overlap information to the local scoring functions can significantly improve their precision and accuracy in a range of resolutions. A combined score involving the local mutual information and overlap (LMI_OV) performs best overall, irrespective of the map category, resolution or the extent of overlap, and we recommend this score for general use. The local mutual information score itself is found to be more discriminatory than cross-correlation coefficient for intermediate-to-low resolution maps or when the map size and density distribution differ significantly. For comparing map surfaces, we implemented two filters to detect the surface points, including one based on the 'extent of surface exposure'. We show that scores that compare surfaces are useful at low resolutions and for maps with evident surface features. All the scores discussed are implemented in TEMPy (http://tempy.ismb.lon.ac.uk/).

Web-based volume slicer for 3D electron-microscopy data from EMDB.

We describe the functionality and design of the Volume slicer - a web-based slice viewer for EMDB entries. This tool uniquely provides the facility to view slices from 3D EM reconstructions along the three orthogonal axes and to rapidly switch between them and navigate through the volume. We have employed multiple rounds of user-experience testing with members of the EM community to ensure that the interface is easy and intuitive to use and the information provided is relevant. The impetus to develop the Volume slicer has been calls from the EM community to provide web-based interactive visualisation of 2D slice data. This would be useful for quick initial checks of the quality of a reconstruction. Again in response to calls from the community, we plan to further develop the Volume slicer into a fully-fledged Volume browser that provides integrated visualisation of EMDB and PDB entries from the molecular to the cellular scale.

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.

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.

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.

Web-based visualisation and analysis of 3D electron-microscopy data from EMDB and PDB.

The Protein Data Bank in Europe (PDBe) has developed web-based tools for the visualisation and analysis of 3D electron microscopy (3DEM) structures in the Electron Microscopy Data Bank (EMDB) and Protein Data Bank (PDB). The tools include: (1) a volume viewer for 3D visualisation of maps, tomograms and models, (2) a slice viewer for inspecting 2D slices of tomographic reconstructions, and (3) visual analysis pages to facilitate analysis and validation of maps, tomograms and models. These tools were designed to help non-experts and experts alike to get some insight into the content and assess the quality of 3DEM structures in EMDB and PDB without the need to install specialised software or to download large amounts of data from these archives. The technical challenges encountered in developing these tools, as well as the more general considerations when making archived data available to the user community through a web interface, are discussed.

OMERO: flexible, model-driven data management for experimental biology.

Data-intensive research depends on tools that manage multidimensional, heterogeneous datasets. We built OME Remote Objects (OMERO), a software platform that enables access to and use of a wide range of biological data. OMERO uses a server-based middleware application to provide a unified interface for images, matrices and tables. OMERO's design and flexibility have enabled its use for light-microscopy, high-content-screening, electron-microscopy and even non-image-genotype data. OMERO is open-source software, available at http://openmicroscopy.org/.

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.

PDBe: Protein Data Bank in Europe.

The Protein Data Bank in Europe (PDBe; pdbe.org) is actively involved in managing the international archive of biomacromolecular structure data as one of the partners in the Worldwide Protein Data Bank (wwPDB; wwpdb.org). PDBe also develops new tools to make structural data more widely and more easily available to the biomedical community. PDBe has developed a browser to access and analyze the structural archive using classification systems that are familiar to chemists and biologists. The PDBe web pages that describe individual PDB entries have been enhanced through the introduction of plain-English summary pages and iconic representations of the contents of an entry (PDBprints). In addition, the information available for structures determined by means of NMR spectroscopy has been expanded. Finally, the entire web site has been redesigned to make it substantially easier to use for expert and novice users alike. PDBe works closely with other teams at the European Bioinformatics Institute (EBI) and in the international scientific community to develop new resources with value-added information. The SIFTS initiative is an example of such a collaboration--it provides extensive mapping data between proteins whose structures are available from the PDB and a host of other biomedical databases. SIFTS is widely used by major bioinformatics resources.