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.

CEITEC Central European Institute of Technology Masaryk University Brno Kamenice 5 625 00 Brno Czech Republic

National Centre for Biomolecular Research Faculty of Science Masaryk University Kotlářská 2 611 37 Brno Czech Republic

Regional Centre of Advanced Technologies and Materials Department of Physical Chemistry Faculty of Science Palacký University tr 17 Listopadu 12 771 46 Olomouc Czech Republic

Regional Centre of Advanced Technologies and Materials Department of Physical Chemistry Faculty of Science Palacký University tř 17 Listopadu 12 771 46 Olomouc Czech Republic

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Analysis and Visualization of Protein Channels, Tunnels, and Pores with MOLEonline and ChannelsDB 2.0

ChannelsDB 2.0: a comprehensive database of protein tunnels and pores in AlphaFold era

Caver Web 1.0: identification of tunnels and channels in proteins and analysis of ligand transport