Large biomolecular structures are being determined experimentally on a daily basis using established techniques such as crystallography and electron microscopy. In addition, emerging integrative or hybrid methods (I/HM) are producing structural models of huge macromolecular machines and assemblies, sometimes containing 100s of millions of non-hydrogen atoms. The performance requirements for visualization and analysis tools delivering these data are increasing rapidly. Significant progress in developing online, web-native three-dimensional (3D) visualization tools was previously accomplished with the introduction of the LiteMol suite and NGL Viewers. Thereafter, Mol* development was jointly initiated by PDBe and RCSB PDB to combine and build on the strengths of LiteMol (developed by PDBe) and NGL (developed by RCSB PDB). The web-native Mol* Viewer enables 3D visualization and streaming of macromolecular coordinate and experimental data, together with capabilities for displaying structure quality, functional, or biological context annotations. High-performance graphics and data management allows users to simultaneously visualise up to hundreds of (superimposed) protein structures, stream molecular dynamics simulation trajectories, render cell-level models, or display huge I/HM structures. It is the primary 3D structure viewer used by PDBe and RCSB PDB. It can be easily integrated into third-party services. Mol* Viewer is open source and freely available at https://molstar.org/.

CATH (https://www.cathdb.info) identifies domains in protein structures from wwPDB and classifies these into evolutionary superfamilies, thereby providing structural and functional annotations. There are two levels: CATH-B, a daily snapshot of the latest domain structures and superfamily assignments, and CATH+, with additional derived data, such as predicted sequence domains, and functionally coherent sequence subsets (Functional Families or FunFams). The latest CATH+ release, version 4.3, significantly increases coverage of structural and sequence data, with an addition of 65,351 fully-classified domains structures (+15%), providing 500 238 structural domains, and 151 million predicted sequence domains (+59%) assigned to 5481 superfamilies. The FunFam generation pipeline has been re-engineered to cope with the increased influx of data. Three times more sequences are captured in FunFams, with a concomitant increase in functional purity, information content and structural coverage. FunFam expansion increases the structural annotations provided for experimental GO terms (+59%). We also present CATH-FunVar web-pages displaying variations in protein sequences and their proximity to known or predicted functional sites. We present two case studies (1) putative cancer drivers and (2) SARS-CoV-2 proteins. Finally, we have improved links to and from CATH including SCOP, InterPro, Aquaria and 2DProt.

BackgroundChannels and pores in biomacromolecules (proteins, nucleic acids and their complexes) play significant biological roles, e.g., in molecular recognition and enzyme substrate specificity.ResultsWe present an advanced software tool entitled MOLE 2.0, which has been designed to analyze molecular channels and pores. Benchmark tests against other available software tools showed that MOLE 2.0 is by comparison quicker, more robust and more versatile. As a new feature, MOLE 2.0 estimates physicochemical properties of the identified channels, i.e., hydropathy, hydrophobicity, polarity, charge, and mutability. We also assessed the variability in physicochemical properties of eighty X-ray structures of two members of the cytochrome P450 superfamily.ConclusionEstimated physicochemical properties of the identified channels in the selected biomacromolecules corresponded well with the known functions of the respective channels. Thus, the predicted physicochemical properties may provide useful information about the potential functions of identified channels. The MOLE 2.0 software is available at http://mole.chemi.muni.cz.

MOLEonline is an interactive, web-based application for the detection and characterization of channels (pores and tunnels) within biomacromolecular structures. The updated version of MOLEonline overcomes limitations of the previous version by incorporating the recently developed LiteMol Viewer visualization engine and providing a simple, fully interactive user experience. The application enables two modes of calculation: one is dedicated to the analysis of channels while the other was specifically designed for transmembrane pores. As the application can use both PDB and mmCIF formats, it can be leveraged to analyze a wide spectrum of biomacromolecular structures, e.g. stemming from NMR, X-ray and cryo-EM techniques. The tool is interconnected with other bioinformatics tools (e.g., PDBe, CSA, ChannelsDB, OPM, UniProt) to help both setup and the analysis of acquired results. MOLEonline provides unprecedented analytics for the detection and structural characterization of channels, as well as information about their numerous physicochemical features. Here we present the application of MOLEonline for structural analyses of α-hemolysin and transient receptor potential mucolipin 1 (TRMP1) pores. The MOLEonline application is freely available via the Internet at https://mole.upol.cz.

PDBsum, http://www.ebi.ac.uk/pdbsum, is a website providing numerous pictorial analyses of each entry in the Protein Data Bank. It portrays the structural features of all proteins, DNA and ligands in the entry, as well as depicting the interactions between them. The latest features, described here, include annotation of human protein sequences with their naturally occurring amino acid variants, dynamic graphs showing the relationships between related protein domain architectures, analyses of ligand binding clusters across different experimental determinations of the same protein, analyses of tunnels in proteins and new search options.

Cytochrome P450 2C9 (CYP2C9) is a membrane-anchored human microsomal protein involved in the drug metabolism in liver. CYP2C9 consists of an N-terminal transmembrane anchor and a catalytic cytoplasmic domain. While the structure of the catalytic domain is well-known from X-ray experiments, the complete structure and its incorporation into the membrane remains unsolved. We constructed an atomistic model of complete CYP2C9 in a dioleoylphosphatidylcholine membrane and evolved it by molecular dynamics simulations in explicit water on a 100+ ns time-scale. The model agrees well with known experimental data about membrane positioning of cytochromes P450. The entry to the substrate access channel is proposed to be facing the membrane interior while the exit of the product egress channel is situated above the interface pointing toward the water phase. The positions of openings of the substrate access and product egress channels correspond to free energy minima of CYP2C9 substrate ibuprofen and its metabolite in the membrane, respectively.

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Human cytochromes P450 (CYPs) are membrane-anchored enzymes involved in biotransformation of many marketed drugs. We constructed atomic models of six human CYPs (CYP1A2, 2A6, 2C9, 2D6, 2E1, and 3A4) anchored to a lipid bilayer to investigate the positions and orientations of CYPs on a membrane. We equilibrated the models by molecular dynamics simulations on a 100+ ns time scale. Catalytic domains of all studied CYPs were found to be partially immersed in the lipid bilayer, whereas the N-terminal part and F'/G' loop are deeply immersed. The proximal side of the enzyme faces the cytosol, whereas the distal side, where openings of substrate access and product release channels to the active site are primarily located, points toward the lipid bilayer. Access channels with openings in the vicinity of the B/C and F/G loops are typically positioned below the lipid head groups, whereas the solvent channel points toward the membrane-water interface. We found that the access channel opening positions match the preferred substrate positions, whereas the product release channel exit positions correspond closely with the positions of the products. This may indicate that membrane-anchored CYPs have evolutionarily adapted to facilitate uptake of nonpolar substrates from the membrane and uptake/release of polar substrates or products from/to the membrane-water interface.