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.

UCSF ChimeraX: Tools for structure building and analysis.

Advances in computational tools for atomic model building are leading to accurate models of large molecular assemblies seen in electron microscopy, often at challenging resolutions of 3-4 Å. We describe new methods in the UCSF ChimeraX molecular modeling package that take advantage of machine-learning structure predictions, provide likelihood-based fitting in maps, and compute per-residue scores to identify modeling errors. Additional model-building tools assist analysis of mutations, post-translational modifications, and interactions with ligands. We present the latest ChimeraX model-building capabilities, including several community-developed extensions. ChimeraX is available free of charge for noncommercial use at https://www.rbvi.ucsf.edu/chimerax.

UCSF ChimeraX: Structure visualization for researchers, educators, and developers.

UCSF ChimeraX is the next-generation interactive visualization program from the Resource for Biocomputing, Visualization, and Informatics (RBVI), following UCSF Chimera. ChimeraX brings (a) significant performance and graphics enhancements; (b) new implementations of Chimera's most highly used tools, many with further improvements; (c) several entirely new analysis features; (d) support for new areas such as virtual reality, light-sheet microscopy, and medical imaging data; (e) major ease-of-use advances, including toolbars with icons to perform actions with a single click, basic "undo" capabilities, and more logical and consistent commands; and (f) an app store for researchers to contribute new tools. ChimeraX includes full user documentation and is free for noncommercial use, with downloads available for Windows, Linux, and macOS from https://www.rbvi.ucsf.edu/chimerax.

Federating Structural Models and Data: Outcomes from A Workshop on Archiving Integrative Structures.

Structures of biomolecular systems are increasingly computed by integrative modeling. In this approach, a structural model is constructed by combining information from multiple sources, including varied experimental methods and prior models. In 2019, a Workshop was held as a Biophysical Society Satellite Meeting to assess progress and discuss further requirements for archiving integrative structures. The primary goal of the Workshop was to build consensus for addressing the challenges involved in creating common data standards, building methods for federated data exchange, and developing mechanisms for validating integrative structures. The summary of the Workshop and the recommendations that emerged are presented here.

Clinical Persistence of Chlamydia trachomatis Sexually Transmitted Strains Involves Novel Mutations in the Functional αßßα Tetramer of the Tryptophan Synthase Operon.

Clinical persistence of Chlamydia trachomatis (Ct) sexually transmitted infections (STIs) is a major public health concern. In vitro persistence is known to develop through interferon gamma (IFN-γ) induction of indoleamine 2,3-dioxygenase (IDO), which catabolizes tryptophan, an essential amino acid for Ct replication. The organism can recover from persistence by synthesizing tryptophan from indole, a substrate for the enzyme tryptophan synthase. The majority of Ct strains, except for reference strain B/TW-5/OT, contain an operon comprised of α and ß subunits that encode TrpA and TrpB, respectively, and form a functional αßßα tetramer. However, trpA mutations in ocular Ct strains, which are responsible for the blinding eye disease known as trachoma, abrogate tryptophan synthesis from indole. We examined serial urogenital samples from a woman who had recurrent Ct infections over 4 years despite antibiotic treatment. The Ct isolates from each infection episode were genome sequenced and analyzed for phenotypic, structural, and functional characteristics. All isolates contained identical mutations in trpA and developed aberrant bodies within intracellular inclusions, visualized by transmission electron microscopy, even when supplemented with indole following IFN-γ treatment. Each isolate displayed an altered αßßα structure, could not synthesize tryptophan from indole, and had significantly lower trpBA expression but higher intracellular tryptophan levels compared with those of reference Ct strain F/IC-Cal3. Our data indicate that emergent mutations in the tryptophan operon, which were previously thought to be restricted only to ocular Ct strains, likely resulted in in vivo persistence in the described patient and represents a novel host-pathogen adaptive strategy for survival.IMPORTANCEChlamydia trachomatis (Ct) is the most common sexually transmitted bacterium with more than 131 million cases occurring annually worldwide. Ct infections are often asymptomatic, persisting for many years despite treatment. In vitro recovery from persistence occurs when indole is utilized by the organism's tryptophan synthase to synthesize tryptophan, an essential amino acid for replication. Ocular but not urogenital Ct strains contain mutations in the synthase that abrogate tryptophan synthesis. Here, we discovered that the genomes of serial isolates from a woman with recurrent, treated Ct STIs over many years were identical with a novel synthase mutation. This likely allowed long-term in vivo persistence where active infection resumed only when tryptophan became available. Our findings indicate an emerging adaptive host-pathogen evolutionary strategy for survival in the urogenital tract that will prompt the field to further explore chlamydial persistence, evaluate the genetics of mutant Ct strains and fitness within the host, and their implications for disease pathogenesis.

Molecular Visualization on the Holodeck.

Can virtual reality be useful for visualizing and analyzing molecular structures and three-dimensional (3D) microscopy? Uses we are exploring include studies of drug binding to proteins and the effects of mutations, building accurate atomic models in electron microscopy and x-ray density maps, understanding how immune system cells move using 3D light microscopy, and teaching schoolchildren about biomolecules that are the machinery of life. Virtual reality (VR) offers immersive display with a wide field of view and head tracking for better perception of molecular architectures and uses 6-degree-of-freedom hand controllers for simple manipulation of 3D data. Conventional computer displays with trackpad, mouse and keyboard excel at two-dimensional tasks such as writing and studying research literature, uses for which VR technology is at present far inferior. Adding VR to the conventional computing environment could improve 3D capabilities if new user-interface problems can be solved. We have developed three VR applications: ChimeraX for analyzing molecular structures and electron and light microscopy data, AltPDB for collaborative discussions around atomic models, and Molecular Zoo for teaching young students characteristics of biomolecules. Investigations over three decades have produced an extensive literature evaluating the potential of VR in research and education. Consumer VR headsets are now affordable to researchers and educators, allowing direct tests of whether the technology is valuable in these areas. We survey here advantages and disadvantages of VR for molecular biology in the context of affordable and dramatically more powerful VR and graphics hardware than has been available in the past.

UCSF ChimeraX: Meeting modern challenges in visualization and analysis.

UCSF ChimeraX is next-generation software for the visualization and analysis of molecular structures, density maps, 3D microscopy, and associated data. It addresses challenges in the size, scope, and disparate types of data attendant with cutting-edge experimental methods, while providing advanced options for high-quality rendering (interactive ambient occlusion, reliable molecular surface calculations, etc.) and professional approaches to software design and distribution. This article highlights some specific advances in the areas of visualization and usability, performance, and extensibility. ChimeraX is free for noncommercial use and is available from http://www.rbvi.ucsf.edu/chimerax/ for Windows, Mac, and Linux.

Actin-based protrusions of migrating neutrophils are intrinsically lamellar and facilitate direction changes.

Leukocytes and other amoeboid cells change shape as they move, forming highly dynamic, actin-filled pseudopods. Although we understand much about the architecture and dynamics of thin lamellipodia made by slow-moving cells on flat surfaces, conventional light microscopy lacks the spatial and temporal resolution required to track complex pseudopods of cells moving in three dimensions. We therefore employed lattice light sheet microscopy to perform three-dimensional, time-lapse imaging of neutrophil-like HL-60 cells crawling through collagen matrices. To analyze three-dimensional pseudopods we: (i) developed fluorescent probe combinations that distinguish cortical actin from dynamic, pseudopod-forming actin networks, and (ii) adapted molecular visualization tools from structural biology to render and analyze complex cell surfaces. Surprisingly, three-dimensional pseudopods turn out to be composed of thin (<0.75 µm), flat sheets that sometimes interleave to form rosettes. Their laminar nature is not templated by an external surface, but likely reflects a linear arrangement of regulatory molecules. Although we find that Arp2/3-dependent pseudopods are dispensable for three-dimensional locomotion, their elimination dramatically decreases the frequency of cell turning, and pseudopod dynamics increase when cells change direction, highlighting the important role pseudopods play in pathfinding.

Biocuration in the structure-function linkage database: the anatomy of a superfamily.

With ever-increasing amounts of sequence data available in both the primary literature and sequence repositories, there is a bottleneck in annotating molecular function to a sequence. This article describes the biocuration process and methods used in the structure-function linkage database (SFLD) to help address some of the challenges. We discuss how the hierarchy within the SFLD allows us to infer detailed functional properties for functionally diverse enzyme superfamilies in which all members are homologous, conserve an aspect of their chemical function and have associated conserved structural features that enable the chemistry. Also presented is the Enzyme Structure-Function Ontology (ESFO), which has been designed to capture the relationships between enzyme sequence, structure and function that underlie the SFLD and is used to guide the biocuration processes within the SFLD. Database URL: http://sfld.rbvi.ucsf.edu/.

An Atlas of Peroxiredoxins Created Using an Active Site Profile-Based Approach to Functionally Relevant Clustering of Proteins.

Peroxiredoxins (Prxs or Prdxs) are a large protein superfamily of antioxidant enzymes that rapidly detoxify damaging peroxides and/or affect signal transduction and, thus, have roles in proliferation, differentiation, and apoptosis. Prx superfamily members are widespread across phylogeny and multiple methods have been developed to classify them. Here we present an updated atlas of the Prx superfamily identified using a novel method called MISST (Multi-level Iterative Sequence Searching Technique). MISST is an iterative search process developed to be both agglomerative, to add sequences containing similar functional site features, and divisive, to split groups when functional site features suggest distinct functionally-relevant clusters. Superfamily members need not be identified initially-MISST begins with a minimal representative set of known structures and searches GenBank iteratively. Further, the method's novelty lies in the manner in which isofunctional groups are selected; rather than use a single or shifting threshold to identify clusters, the groups are deemed isofunctional when they pass a self-identification criterion, such that the group identifies itself and nothing else in a search of GenBank. The method was preliminarily validated on the Prxs, as the Prxs presented challenges of both agglomeration and division. For example, previous sequence analysis clustered the Prx functional families Prx1 and Prx6 into one group. Subsequent expert analysis clearly identified Prx6 as a distinct functionally relevant group. The MISST process distinguishes these two closely related, though functionally distinct, families. Through MISST search iterations, over 38,000 Prx sequences were identified, which the method divided into six isofunctional clusters, consistent with previous expert analysis. The results represent the most complete computational functional analysis of proteins comprising the Prx superfamily. The feasibility of this novel method is demonstrated by the Prx superfamily results, laying the foundation for potential functionally relevant clustering of the universe of protein sequences.

An approach to functionally relevant clustering of the protein universe: Active site profile-based clustering of protein structures and sequences.

Protein function identification remains a significant problem. Solving this problem at the molecular functional level would allow mechanistic determinant identification-amino acids that distinguish details between functional families within a superfamily. Active site profiling was developed to identify mechanistic determinants. DASP and DASP2 were developed as tools to search sequence databases using active site profiling. Here, TuLIP (Two-Level Iterative clustering Process) is introduced as an iterative, divisive clustering process that utilizes active site profiling to separate structurally characterized superfamily members into functionally relevant clusters. Underlying TuLIP is the observation that functionally relevant families (curated by Structure-Function Linkage Database, SFLD) self-identify in DASP2 searches; clusters containing multiple functional families do not. Each TuLIP iteration produces candidate clusters, each evaluated to determine if it self-identifies using DASP2. If so, it is deemed a functionally relevant group. Divisive clustering continues until each structure is either a functionally relevant group member or a singlet. TuLIP is validated on enolase and glutathione transferase structures, superfamilies well-curated by SFLD. Correlation is strong; small numbers of structures prevent statistically significant analysis. TuLIP-identified enolase clusters are used in DASP2 GenBank searches to identify sequences sharing functional site features. Analysis shows a true positive rate of 96%, false negative rate of 4%, and maximum false positive rate of 4%. F-measure and performance analysis on the enolase search results and comparison to GEMMA and SCI-PHY demonstrate that TuLIP avoids the over-division problem of these methods. Mechanistic determinants for enolase families are evaluated and shown to correlate well with literature results.

DASP3: identification of protein sequences belonging to functionally relevant groups.

BACKGROUND: Development of automatable processes for clustering proteins into functionally relevant groups is a critical hurdle as an increasing number of sequences are deposited into databases. Experimental function determination is exceptionally time-consuming and can't keep pace with the identification of protein sequences. A tool, DASP (Deacon Active Site Profiler), was previously developed to identify protein sequences with active site similarity to a query set. Development of two iterative, automatable methods for clustering proteins into functionally relevant groups exposed algorithmic limitations to DASP. RESULTS: The accuracy and efficiency of DASP was significantly improved through six algorithmic enhancements implemented in two stages: DASP2 and DASP3. Validation demonstrated DASP3 provides greater score separation between true positives and false positives than earlier versions. In addition, DASP3 shows similar performance to previous versions in clustering protein structures into isofunctional groups (validated against manual curation), but DASP3 gathers and clusters protein sequences into isofunctional groups more efficiently than DASP and DASP2. CONCLUSIONS: DASP algorithmic enhancements resulted in improved efficiency and accuracy of identifying proteins that contain active site features similar to those of the query set. These enhancements provide incremental improvement in structure database searches and initial sequence database searches; however, the enhancements show significant improvement in iterative sequence searches, suggesting DASP3 is an appropriate tool for the iterative processes required for clustering proteins into isofunctional groups.

Outcome of the First wwPDB Hybrid/Integrative Methods Task Force Workshop.

Structures of biomolecular systems are increasingly computed by integrative modeling that relies on varied types of experimental data and theoretical information. We describe here the proceedings and conclusions from the first wwPDB Hybrid/Integrative Methods Task Force Workshop held at the European Bioinformatics Institute in Hinxton, UK, on October 6 and 7, 2014. At the workshop, experts in various experimental fields of structural biology, experts in integrative modeling and visualization, and experts in data archiving addressed a series of questions central to the future of structural biology. How should integrative models be represented? How should the data and integrative models be validated? What data should be archived? How should the data and models be archived? What information should accompany the publication of integrative models?

Multidomain Assembler (MDA) Generates Models of Large Multidomain Proteins.

Homology modeling predicts protein structures using known structures of related proteins as templates. We developed MULTIDOMAIN ASSEMBLER (MDA) to address the special problems that arise when modeling proteins with large numbers of domains, such as fibronectin with 30 domains, as well as cases with hundreds of templates. These problems include how to spatially arrange nonoverlapping template structures, and how to get the best template coverage when some sequence regions have hundreds of available structures while other regions have a few distant homologs. MDA automates the tasks of template searching, visualization, and selection followed by multidomain model generation, and is part of the widely used molecular graphics package UCSF CHIMERA (University of California, San Francisco). We demonstrate applications and discuss MDA's benefits and limitations.

cddApp: a Cytoscape app for accessing the NCBI conserved domain database.

MOTIVATION: cddApp is a Cytoscape extension that supports the annotation of protein networks with information about domains and specific functional sites from the National Center for Biotechnology Information's conserved domain database (CDD). CDD information is loaded for nodes annotated with NCBI numbers or UniProt identifiers and (optionally) Protein Data Bank structures. cddApp integrates with the Cytoscape apps structureViz2 and enhancedGraphics. Together, these three apps provide powerful tools to annotate nodes with CDD domain and site information and visualize that information in both network and structural contexts. AVAILABILITY AND IMPLEMENTATION: cddApp is written in Java and freely available for download from the Cytoscape app store (http://apps.cytoscape.org). Documentation is provided at http://www.rbvi.ucsf.edu/cytoscape, and the source is publically available from GitHub http://github.com/RBVI/cddApp.

RRDistMaps: a UCSF Chimera tool for viewing and comparing protein distance maps.

MOTIVATION: Contact maps are a convenient method for the structural biologists to identify structural features through two-dimensional simplification. Binary (yes/no) contact maps with a single cutoff distance can be generalized to show continuous distance ranges. We have developed a UCSF Chimera tool, RRDistMaps, to compute such generalized maps in order to analyze pairwise variations in intramolecular contacts. An interactive utility, RRDistMaps, visualizes conformational changes, both local (e.g. binding-site residues) and global (e.g. hinge motion), between unbound and bound proteins through distance patterns. Users can target residue pairs in RRDistMaps for further navigation in Chimera. The interface contains the unique features of identifying long-range residue motion and aligning sequences to simultaneously compare distance maps. AVAILABILITY AND IMPLEMENTATION: RRDistMaps was developed as part of UCSF Chimera release 1.10, which is freely available at http://rbvi.ucsf.edu/chimera/download.html, and operates on Linux, Windows, and Mac OS. CONTACT: conrad@cgl.ucsf.edu.

CyAnimator: Simple Animations of Cytoscape Networks.

CyAnimator (http://apps.cytoscape.org/apps/cyanimator) is a Cytoscape app that provides a tool for simple animations of Cytoscape networks. The tool allows you to take a series of snapshots (CyAnimator calls them frames) of Cytoscape networks. For example, the first frame might be of a network shown from a "zoomed out" viewpoint and the second frame might focus on a specific group of nodes. Once these two frames are captured by the tool, it can animate between them by interpolating the changes in location, zoom, node color, node size, edge thickness, presence or absence of annotations, etc. The animations may be saved as a series of individual frames, animated GIFs, MP4 movies, or H.264/MOV movies. CyAnimator is available from within the Cytoscape App Manager or from the Cytoscape app store.

enhancedGraphics: a Cytoscape app for enhanced node graphics.

enhancedGraphics ( http://apps.cytoscape.org/apps/enhancedGraphics) is a Cytoscape app that implements a series of enhanced charts and graphics that may be added to Cytoscape nodes. It enables users and other app developers to create pie, line, bar, and circle plots that are driven by columns in the Cytoscape Node Table. Charts are drawn using vector graphics to allow full-resolution scaling.

Enhancing UCSF Chimera through web services.

Integrating access to web services with desktop applications allows for an expanded set of application features, including performing computationally intensive tasks and convenient searches of databases. We describe how we have enhanced UCSF Chimera (http://www.rbvi.ucsf.edu/chimera/), a program for the interactive visualization and analysis of molecular structures and related data, through the addition of several web services (http://www.rbvi.ucsf.edu/chimera/docs/webservices.html). By streamlining access to web services, including the entire job submission, monitoring and retrieval process, Chimera makes it simpler for users to focus on their science projects rather than data manipulation. Chimera uses Opal, a toolkit for wrapping scientific applications as web services, to provide scalable and transparent access to several popular software packages. We illustrate Chimera's use of web services with an example workflow that interleaves use of these services with interactive manipulation of molecular sequences and structures, and we provide an example Python program to demonstrate how easily Opal-based web services can be accessed from within an application. Web server availability: http://webservices.rbvi.ucsf.edu/opal2/dashboard?command=serviceList.