PDB: Deposited Structures as Functional-Insight Evidence

Goal

Many genes in this review pipeline have experimentally solved 3D structures deposited in
the Protein Data Bank. A structure is not just a picture: when it
captures a bound ligand, cofactor, catalytic metal, or partner macromolecule, it is
direct, experimental evidence for molecular function, binding, and complex membership — the
very things GO annotation review hinges on.

This project (1) inventories what is deposited across the whole pipeline, and (2) prioritizes
the structures most likely to change or strengthen a gene's functional annotation, so that
structure-informed review effort goes where it pays off.

What "functional insight" means here

A deposited structure is most valuable for review when what is bound in it tells us
something the current annotations do not. The prioritization therefore combines two axes:

1. Structure richness — what the structure actually contains:
2. bound cofactor / catalytic metal (FAD, NAD(P), PQQ, F430, Fe–S clusters, heme, Zn, …)
   → strongest single clue to catalytic mechanism / molecular function
3. bound substrate / product / inhibitor ligand → active-site and specificity evidence
4. complex (≥2 protein entities, or bound nucleic acid) → interaction & CC evidence

5. high sequence coverage (full-length vs a short peptide fragment in a partner's structure)

6. Annotation sparsity — a structure adds the most where we currently know the least.
   Genes whose GO annotations are electronic-only (IEA/IBA) with no experimental
   molecular-function term are upweighted: here a ligand-bound structure (plus literature)
   can ground a specific, experiment-grade MF annotation that today rests only on inference.

The two are multiplied: richness × sparsity. An apo structure of an already
well-annotated protein scores low; a cofactor-bound, full-length structure of an IEA-only
enzyme scores high.

What is deposited (pipeline-wide inventory)

Computed offline from the cached UniProt records (*-uniprot.txt, DR PDB cross-references):

Top organisms by genes-with-structure: human (588), yeast (65), ARATH (53), BPT4 (32),
mouse (30), ECOLI (23), PSEPK (19), SCHPO (17), worm (17), rat (15).

Three prioritization cuts

enrich_rcsb.py enriches the union of three candidate cuts (278 genes; each tagged in the
candidate_reason column), so the priority list is no longer prokaryote-dominated:

Full inventory: PDB/data/pdb_inventory.tsv (per entry) and
pdb_gene_summary.tsv (per gene).

Prioritized candidates (first pass)

The 114 genes with a structure but no experimental MF GO were enriched with RCSB metadata
(bound ligands, entity counts, titles). Of the 105 that resolved:

• 42 have a structure with a bound cofactor / catalytic metal
• 69 have a structure with a meaningful (non-buffer) ligand
• 40 are captured in a complex; 6 with bound nucleic acid
• 23 are apo with no partner (lower structural-insight yield)

These are IEA-only enzymes/proteins whose function is structurally (and often
biochemically) characterized but whose GO annotations have not been promoted beyond
electronic inference — the sweet spot for structure-grounded review.

Full ranked list: PDB/data/pdb_gene_enriched.tsv (now includes the
RCSB per-entry structure-paper PMIDs and an is_eukaryote flag).

Eukaryotic candidates (so they aren't drowned out)

Broadening beyond the strict "no experimental MF" cut to euk + contested surfaces a
much richer eukaryotic slice (815 eukaryotic genes have a structure). Top eukaryotic
candidates by score, with the cut(s) that flagged them:

The verified flagships (IDH3B, ATAD1, XYL1, psaC, COX6B1, SPR, COI1) are written up in
PDB/STRUCTURE_PAPERS.md. For human genes the structure typically sharpens an
existing IEA term or pins complex membership rather than revealing function from scratch.

Contested catalytic functions (structure adjudicates)

130 genes with a structure have a review that marked a catalytic molecular function as
REMOVE or over-annotated. A deposited structure is decisive here — it shows whether the
cofactor/active-site pocket is actually present. Two distinct cases (don't conflate them):

• Over-general parent marked over-annotated (e.g. DOT1/SIRT2 "transferase activity",
  XYL1/pobA "oxidoreductase activity"): the bound cofactor confirms catalysis and the
  structure points to the specific child term that should replace the generic one.
• Genuinely-wrong specific activity marked REMOVE (e.g. ARATH HEN1 "peptidyl-prolyl
  isomerase" — but bound SAH argues for its real RNA-methyltransferase activity; human
  CASP3 tagged "aspartic-type endopeptidase" when it is a cysteine protease; BRCA2
  "histone acetyltransferase"): the structure/cofactor adjudicates against the wrong call.

Full list with all disputed terms per gene: pdb_gene_enriched.tsv (candidate_reason
contains contested; contested_cat_mf lists the term/label/action). As always, the
disputed-term mapping and the structure PMID must both be verified before use.

Grounding in the structure papers

A bound ligand is the clue; the primary structure paper carries the functional
interpretation. PDB/STRUCTURE_PAPERS.md records verified, PubMed-sourced
notes for the shortlist above (and the prokaryotic flagships mcrA, merA, pcaF), with the
GO-annotation implication for each.

Caveat surfaced by doing this: the structure_papers PMIDs are the RCSB per-entry
primary citation — the paper that deposited that coordinate set, which is often a downstream
ligand/inhibitor or methods study rather than the definitive structure/function paper. Verified
drift cases: SPR's PMIDs are inhibitor-screening papers; cbh1's are glycosylation/propranolol
NMR; merA's is the N-terminal NmerA-domain NMR. Others (XYL1, psaC, IDH3B, ATAD1, pcaF, COX6B1)
are the definitive paper. Each PMID must be verified against the gene before it is cited in
a review (the "verify, don't trust" rule), exactly as STRUCTURE_PAPERS.md does.

Patterns worth noting: a cluster of methylotrophy / PQQ-dependent dehydrogenases
(METEA mxaI, xoxF1, mdh, mtdA, fae, PSEPK pedH, pqqB) and redox cofactor
enzymes (merA FAD/NADP, psaC/mcrA/pqqE Fe–S, DsrAB heme/siroheme) — these have
diagnostic cofactors visible in their structures, making them low-effort, high-yield review
targets.

Reproduce

# 1. offline inventory from cached UniProt + GOA (no network)
python3 projects/PDB/inventory_pdb.py
# 2. RCSB enrichment of the prioritized candidate genes (network)
python3 projects/PDB/enrich_rcsb.py
# 3. structure-paper -> GOA citation gap (offline); writes CURATION_GAP.md
python3 projects/PDB/curation_gap.py
# 4. ranked GAP_OPPORTUNITY review worklist (offline); writes GAP_WORKLIST.md
python3 projects/PDB/gap_worklist.py
# 5. H1 frontier test set: GAP_NO_EXP_CURATION genes (offline); writes data/h1_testset.tsv
python3 projects/PDB/h1_testset.py

See PDB/RESULTS.md for method detail, caveats, and the full output schema.

Does structural evidence fill annotation gaps? (PDB/H1_LEDGER.md)

H1_LEDGER.md tests whether structures fill GO gaps that traditional publications would
not. It records the GAP_OPPORTUNITY→GAP_NO_EXP_CURATION methodology correction, the
three-evidence-layer model of a structure paper (coordinates → low-information binding
terms; the paper's integrative hypothesis → informative function; sequence/EC → catalytic
identity), and the Layer-2 scoring pass. Bottom line: structures reliably supply first
experimental-grade evidence for under-curated proteins, but genuinely new informative
function is rare — throttled by subunit mismatch and GO expressivity.

Caveats

• The "no experimental MF GO" flag is computed from the cached *-goa.tsv. It flags genes
  whose GO is electronic-only in our snapshot; some are well-characterized in the
  literature. That is the point — the structure + literature can justify promoting the
  annotation, not that the function is unknown.
• A bound ligand is a clue, not proof: crystallization additives are filtered out, but a
  flagged ligand still needs reviewer judgment (substrate vs adventitious binder).
• UniProt DR PDB residue ranges drive the coverage metric; a gene present only as a short
  peptide in a partner's structure scores low coverage, correctly.

Are structure papers overlooked by curation? (PDB/CURATION_GAP.md)

PDB/curation_gap.py measures, for every deposited structure with a linked primary
publication, whether that PMID is cited in the gene's GOA REFERENCE column. Across
737 structure-paper × gene pairs (247 genes), only 15% are cited by GOA; 65%
are GAP_OPPORTUNITY (the paper predates the gene's last experimental annotation yet is
never referenced), and 174/247 genes cite zero of their structure papers. "Not cited"
means the structural study is absent from the evidence trail, not that the function is
unannotated — but it quantifies how under-used the structural literature is as a GO evidence
source. The lag boundary uses the latest experimental annotation year, since overall GOA
dates are inflated by IEA/IBA pipeline refreshes.

The reusable GOA-citation helper lives in core
(ai_gene_review.validation.goa_validator.referenced_pmids); the analysis is
project-specific.

Prioritized worklist (PDB/GAP_WORKLIST.md)

PDB/gap_worklist.py ranks the GAP_OPPORTUNITY papers by gene priority
(dark-MF / eukaryote / contested) plus the cofactor / ligand / complex richness of the
uncited structures, collapsed to one row per gene (the review unit). Top targets:
yeast PNO1, human RPS3, human BIRC5, human GCH1, human SIRT2, human MAPK1,
ARATH CRY2. Per-paper detail in PDB/data/gap_worklist.tsv.

Next steps

• [x] First eukaryotic batch reviewed with structure evidence: PICST XYL1, human IDH3B,
  human COX6B1, CHLRE psaC, ARATH COI1, human ATAD1.
• [ ] Work down GAP_WORKLIST.md from the top, citing the verified structure paper + PDB
  entry + bound cofactor as evidence; remaining shortlist includes prokaryotic
  PSEPK pcaF, METAC mcrA, PSEAI merA.
• [ ] Extend enrichment beyond the no-exp-MF set to genes with contested function
  (cross-reference CONTESTED_FUNCTION.md) where a structure could adjudicate.
• [ ] For complexes (≥2 protein entities), map partners to UniProt to support CC / complex
  membership annotations.
• [ ] Replace each peripheral RCSB auto-citation with the definitive structure/function paper
  (see the caveat above) as genes go to review.
• [ ] Consider adding a PDB-evidence field to the review schema (per ALPHAFOLD.md action items).