The RCSB Protein Data Bank (RCSB PDB) has developed a new pairwise structure alignment tool, accessible at rcsb.org/alignment, which integrates seamlessly into the RCSB.org web portal. This tool allows users to align multiple protein structures to a reference structure using established alignment algorithms such as FATCAT, CE, TM-align, or Smith–Waterman 3D. The user-friendly interface simplifies parameter setup and input selection, enabling visualization of results in both 1D sequence and 3D structural perspectives. Users can compare structures from the PDB archive with over a million computed structure models from the ModelArchive and AlphaFold DB. The tool also supports custom structure data via URLs or uploaded files. Alignment results can be bookmarked and shared with collaborators. The tool provides a range of alignment methods, including rigid and flexible alignments, which are particularly useful for partially disordered proteins. It supports various input formats, including PDB, mmCIF, and BinaryCIF. Users can export results in different formats, such as FASTA for sequence alignments and JSON for 3D transformation matrices. The tool also integrates with the RCSB Sequence Annotations Viewer and the Mol* 3D viewer for interactive exploration of alignments at both 1D and 3D levels. The alignment API (alignment.rcsb.org) allows programmatic access, with asynchronous processing for computationally intensive tasks. Each user request is assigned a unique ticket to track progress. The tool is part of the RCSB PDB research-focused RCSB.org web portal and is available for both graphical and programmatic access. It supports data from public URLs and user-uploaded atomic coordinate files, and can reference structures using UniProt, AlphaFold DB, or ESM Metagenomic Atlas identifiers. The tool facilitates comprehensive structural and sequence analysis, enhancing understanding of protein evolutionary relationships.The RCSB Protein Data Bank (RCSB PDB) has developed a new pairwise structure alignment tool, accessible at rcsb.org/alignment, which integrates seamlessly into the RCSB.org web portal. This tool allows users to align multiple protein structures to a reference structure using established alignment algorithms such as FATCAT, CE, TM-align, or Smith–Waterman 3D. The user-friendly interface simplifies parameter setup and input selection, enabling visualization of results in both 1D sequence and 3D structural perspectives. Users can compare structures from the PDB archive with over a million computed structure models from the ModelArchive and AlphaFold DB. The tool also supports custom structure data via URLs or uploaded files. Alignment results can be bookmarked and shared with collaborators. The tool provides a range of alignment methods, including rigid and flexible alignments, which are particularly useful for partially disordered proteins. It supports various input formats, including PDB, mmCIF, and BinaryCIF. Users can export results in different formats, such as FASTA for sequence alignments and JSON for 3D transformation matrices. The tool also integrates with the RCSB Sequence Annotations Viewer and the Mol* 3D viewer for interactive exploration of alignments at both 1D and 3D levels. The alignment API (alignment.rcsb.org) allows programmatic access, with asynchronous processing for computationally intensive tasks. Each user request is assigned a unique ticket to track progress. The tool is part of the RCSB PDB research-focused RCSB.org web portal and is available for both graphical and programmatic access. It supports data from public URLs and user-uploaded atomic coordinate files, and can reference structures using UniProt, AlphaFold DB, or ESM Metagenomic Atlas identifiers. The tool facilitates comprehensive structural and sequence analysis, enhancing understanding of protein evolutionary relationships.