Overview
Database paper: Teixeira et al., bioRxiv (2026) (preprint). See How to cite OpenAntigens.
Each release records the results of a local analysis. Reports link proposed construct boundaries to the sequence, annotations, and structural evidence used in that analysis.
The methods below describe the current pipeline. Previously generated releases retain the results of the software and reference data used for their build.
Target universe and resolution
OpenAntigens Mouse contains unique mouse orthologs resolved from the OpenAntigens human target universe. Targets without a resolved mouse record are omitted, and duplicate mouse records are included once. Most included targets are analyzed against reviewed mouse UniProt data; where no reviewed mouse entry covers the ortholog, the sequence comes from the RefSeq record named in the report's sequence source. The originating human target remains in report metadata.
Mouse records are resolved through reviewed mouse UniProt entries where one exists, and otherwise through the RefSeq sequence in the precomputed ortholog table; each report states which it used. The build records skipped human targets, including missing mouse orthologs and duplicate mouse targets, in a separate manifest.
Topology and construct scope
Topology controls which design track is used. Secreted proteins use the mature extracellular protein as the soluble design scope. GPI-anchored proteins without a conventional transmembrane helix use the envelope of processed UniProt chain annotations. If no processed chain contains the annotated GPI anchor, OpenAntigens retains the post-signal sequence without trimming the GPI propeptide and marks that limitation. Single-pass proteins use the extracellular region defined by topology and transmembrane annotations.
Multipass proteins are treated separately. When an eligible extracellular region has at least 80 amino acids by default, OpenAntigens includes a mixed track with both soluble extracellular-region constructs and full-length membrane-expression context. With shorter extracellular loops, the report emphasizes membrane-protein expression and full-length context.
Sequence canonicality
OpenAntigens requires the report sequence, UniProt sequence, and AlphaFold model sequence to represent the same canonical isoform. Mismatching AlphaFold models are excluded from structure-dependent metrics because residue-number mismatches corrupt construct boundaries, pLDDT coloring, and sequence-to-structure selection.
AlphaFold DB models are used only when they match the canonical report sequence.
When a target lacks a matching AlphaFold DB model, non-structure-dependent annotations remain available; structure-derived segmentation and interactive structure features are limited.
AlphaFold confidence
pLDDT is used as local residue-confidence evidence. High pLDDT supports local fold confidence, while low pLDDT is consistent with flexible linkers, disordered tails, or uncertain loops. PAE is used as regional confidence evidence: low internal PAE supports a coherent structural unit, while high PAE between neighboring blocks indicates uncertain relative orientation.
Construct diagnostics summarize mean pLDDT, structured fraction, mean intrablock PAE, interblock PAE, local boundary PAE, separation, and linker pLDDT when available. These metrics are evidence features, not absolute pass/fail criteria.
Residue topology and accessibility
Residues are classified by topology first, using UniProt topology features, transmembrane segments, signal peptides, propeptides, mature-chain annotations, and the input topology track. The reported location categories are extracellular, membrane, intracellular, or unknown. For multipass proteins, extracellular termini and loops are treated as extracellular regions inside a membrane-protein model.
Surface accessibility is computed from the canonical AlphaFold structure with FreeSASA using the Lee-Richards algorithm. The default probe radius is 1.4 angstroms, the default slice count is 20, and residues are called solvent-accessible when relative SASA is at least 0.20.
Extracellular accessible residues
OpenAntigens combines topology and model accessibility to select extracellular residues for comparison. This set is used in ortholog and family identity calculations, BLAST summaries, and alignment highlighting.
The pipeline also calculates SASA within PAE blocks to inspect residues that may be hidden by uncertain packing between blocks. Recomputing SASA within a block shows how its calculated accessibility changes when other blocks are removed; it does not establish accessibility in solution or on cells.
SASA calculations use the target-chain model and selected structural blocks, without partner chains. The pipeline does not reconstruct biological homo-oligomeric assemblies or incorporate their interfaces into boundary selection. A residue exposed in the model may be buried against another subunit, including an identical copy, in the biological assembly. No solvent-exposure call is made inside a transmembrane or intramembrane span: solvent accessibility is undefined there without a membrane model, and lipid-facing cannot be told from bundle-facing in a monomer. Curated UniProt disulfides take precedence over model geometry when both ends lie in the analyzed range.
The extracellular accessible identity reported in homolog, family, and BLAST sections is calculated only over aligned primary-target residues that are both extracellular and classified as exposed or conditional.
Construct generation
The analysis engine scores construct candidates, removes exact duplicate boundaries across calculated and annotated candidate sources (PDB-backed constructs are kept individually as evidence), filters soluble candidates outside the inferred design scope or below the minimum length, and the portal displays the surviving classes in a fixed review order.
The minimum construct size defaults to 50 amino acids. Soluble constructs outside the inferred design scope are excluded. Exact duplicate boundaries from different calculated or annotated sources are collapsed during construct deduplication; PDB-backed constructs are exempt from deduplication, but still pass the scope and length filters.
The Constructs page gives the default pLDDT thresholds, PAE split criteria, and boundary score. These classes describe the algorithm used, not measured expression or binding.
Domain annotations
UniProt feature annotations and InterPro records are merged into the residue annotation map. Domain annotations must lie inside the relevant design scope to be used as construct recommendations. Large whole-protein signatures that extend beyond the mature secreted or extracellular region are retained as descriptive context when appropriate. They are excluded from soluble construct recommendations.
InterPro provides integrated domain and family annotations. Pfam entries support domain annotation. Pfam-only family IDs are excluded as canonical family identifiers for precomputed family matrices.
Family and paralog context
Canonical family assignment prefers InterPro family annotations when they are specific and informative. HGNC and Ensembl-derived paralog references are used to recover biologically relevant families when InterPro is incomplete or too broad. If multiple families are available, smaller and more specific families are preferred over large generic families.
Large families can be skipped from detailed matrix rendering to keep pages readable. For each globally aligned pair, directional identity divides the number of identical residues by the length of the row protein's sequence region. Reversing the comparison changes the denominator when the lengths differ.
Orthologs and homology
The mouse portal keeps the originating human target in report metadata and maps additional homolog regions when suitable reference sequences are available.
Construct-level homolog sequences are inferred by aligning the mouse design scope to available homolog sequences, projecting the selected mouse boundaries through that alignment, and extracting the corresponding homolog region. A projection is rejected when fewer than 70% of the target-region residues align to the homolog.
Experimental structures
PDB/RCSB evidence is mapped back to UniProt residue numbering where possible. Experimental construct boundaries that overlap the design scope are summarized and can become PDB-backed construct suggestions. PDB entries fully outside the mature secreted, extracellular, or relevant membrane-protein scope are ignored for construct recommendation.
Use the linked PDB record to review the mapped chain and original experiment.
BLAST sequence similarity and cross-reactivity context
Local blastp searches provide sequence similarity context for reviewing possible cross-reactivity. Mouse target sequences are searched against the configured local human, mouse, and cynomolgus monkey protein databases.
Self hits are filtered by accession and entry name. BLAST results are ranked sequence similarity signals, not measurements of antibody cross-reactivity. No universal threshold is applied because acceptable risk depends on antigen class, screening goal, and downstream assay design.
PTMs and processing features
OpenAntigens extracts curated UniProt PTM-like features including glycosylation, modified residues, lipidation, disulfide bonds, cross-links, initiator methionine processing, signal peptides, propeptides, mature chains, peptides, and cleavage-related site annotations.
Target pages list available annotations in a PTM section. Construct cards list which PTMs overlap each construct, and the Interactive Construct Builder updates the included PTMs in real time as the selected residue window changes. Processing annotations are included in boundary warnings.
Cysteine analysis
Model pairing uses cysteine sulfur atoms within 2.4 angstroms. A pair is assigned only when each cysteine has exactly one candidate partner and the choices are reciprocal. Competing contacts are marked ambiguous. Curated UniProt disulfides are shown separately as annotations.
The Interactive Construct Builder can optionally apply Cys-to-Ser edits to selected unpaired cysteines and updates copied TSV/FASTA sequences in real time.
Furin-site scanning
The default scan finds R-X-[K/R]-R motifs, including overlapping matches, in the design region and constructs. The pipeline reports suggested edits as annotations and leaves application to manual review because cleavage risk is context-dependent and some basic motifs may be biologically important.
Ligand and interaction regions
Curated UniProt features, PDB mappings, and annotation text are used to report ligand-binding or interaction-related regions when available. Check these annotations before trimming the sequence.
Assembly and partner requirements
Interaction and complex information is summarized separately from obligatory-partner classification. Known interactions provide context; obligatory-partner warnings require stronger curated complex evidence.
Obligatory-partner warnings are anchored to curated Complex Portal records when that module is enabled. UniProt SUBUNIT comments inform integrin assembly classification but do not independently create obligatory-partner warnings. Other free-text interaction rows are not shown in the public portal.
A listed interaction alone does not produce an obligatory-partner warning. Check the evidence class and source record before deciding whether to include a partner.
Disease, literature, and antibody-resource context
The mouse portal does not display Open Targets disease associations or disease-search controls.
PubTator3 is queried for approximate gene-linked PubMed hit counts. The counts help locate literature; they do not grade its quality or validate a target. CiteAb links are provided as external antibody-resource shortcuts; CiteAb catalog content remains external.
Static assets and visualization
When assets are generated, each construct can include structure images, pLDDT plots, and PAE plots with the construct region highlighted. Structure renderings use AlphaFold models colored by pLDDT and emphasize cysteines when possible. The portal also includes an Interactive Construct Builder that links sequence selection, structure highlighting, pLDDT, PAE, warnings, and copy-ready sequence outputs.
If the interactive builder is unavailable, the precomputed cards still provide sequences and any saved plots.
Update cadence and limitations
OpenAntigens is released as a static, versioned snapshot. Each release records the build date, target universe, and OpenAntigens version when available. Future releases may incorporate updated annotations.
Because each release is static, source databases may have changed after the displayed release date. Cite the release date and downloaded artifact names when using OpenAntigens data in downstream analyses.
