ligD Resolved · high auto-curated

Annotation: from legacy to revised

Auto-curated: this verdict and function were generated by rules from PGAP + Pfam + Foldseek and have not been hand-reviewed.

In the literature (TB corpus sweep) cited only under an ortholog name

Found under: M. smegmatis (1).

1 TB publication mentions this gene. Invisible under its H37Rv locus tag: this gene appears in the literature ONLY under its ortholog identifier(s) (M. smegmatis). Searching 'Rv3730c' alone finds nothing.

This layer CITES the literature, it does not change the verdict or the function. A gene may be heavily studied (as an antigen, a resistance determinant, a drug target) while its molecular function is settled elsewhere in the fiche. This distinguishes a gene that is dark because nobody has looked from one that is dark despite having been studied. Source: PubMed (whole), MULTI-ALIAS sweep: H37Rv locus tag AND every ortholog identifier (M. bovis Mb…, M. marinum MMAR_…, M. smegmatis MSMEG_…, M. leprae ML…, M. abscessus MAB_…), each hit VERIFIED against the abstract text (word-boundary regex). phase73/phase75, 2026-07-13.

CRISPRi vulnerability

Vulnerability index 0.27 (95% CI -3.15 to 5.13). A more negative index = more vulnerable to knockdown (better drug-target quality); indicative threshold VI ≤ -6 = highly vulnerable.

Quantitative CRISPRi knockdown, graded (finer than binary Tn-seq essentiality). Source: CRISPRi vulnerability index (Bosch 2021, supplementary Table S2 'mmc3.xlsx', bulk import via Europe PMC -- supersedes the per-gene pebble.rockefeller.edu scrape of phase46_crispri_vulnerability.py).

Caveat on this estimate. Bosch et al. flag this gene's vulnerability estimate as NOT certain, and here that call has a mechanical cause visible in the library itself: every guide targeting this gene is >=21 nt (str_span=0.261 on a 0-1 scale, near the genome floor), and guide repression strength saturates from 21 nt on -- so the library never sampled a partial knockdown for this gene, only the near-maximal repression plateau. The dose-response fit (and this VI) is therefore essentially unconstrained by data, not merely uncertain -- a limitation of the CRISPRi library design for the non-essential class (Bosch et al. 2021), not a defect in this specific measurement. n_guides=8 is descriptive only and does not predict this. Do not cite this VI as a standalone vulnerability claim.

Legacy record & comparison (Mycobrowser) ahead of Mycobrowser

Mycobrowser classes this locus among conserved hypotheticals; the atlas now assigns a functional handle (COG category, requalified function). Mycobrowser is no longer maintained, so its EC numbers predate recent nomenclature revisions (e.g. the 2018 EC 7 "translocase" class) — most EC differences are re-numberings of the same enzyme, not conflicts.

Orthologues (reciprocal best hits across mycobacteria)

Reciprocal-best-hit orthologues (DIAMOND) against the Mycobrowser reference proteomes. A missing species is informative: e.g. a gene absent from M. leprae was likely lost in its reductive genome evolution. Locus tags link to Mycobrowser.

Curated reference (UniProt)

Functional vocabulary (eggNOG-mapper, orthology transfer)

Orthology-based transfer (eggNOG 5.0.2, diamond). EC/KO/GO/CAZy are computed annotations, not manual curation; cross-check against the primary literature before treating a specific reaction as established.

Conservation & selection (intra-MTBC, 142 601 strains, snapshot of 2026-09-29)

pN/pS from segregating SNPs (singletons removed) normalised by possible sites. Low pN/pS = purifying selection (a strong signal that a "hypothetical" is a real, constrained gene). A high pN/pS is ambiguous: relaxed constraint or positive selection (drug resistance, antigenic variation) inflate it; e.g. rpoB/katG/pncA score high here for resistance, not loss of function. A clonal disruption (one allele over a clade) suggests lineage pseudogenisation; a convergent one (many independent alleles) is typical of resistance loss-of-function.

Outgroup conservation (beyond the MTBC) Actinomycetia

Two orthogonal outgroup signals. M. canettii (the immediate outgroup) gives a deep-divergence dN/dS (a low value confirms a constrained, real gene; shown as confident only at ≥8 substitutions, else flagged low-power). Genus-wide presence/absence (tblastn vs assembled non-MTBC genomes) places the gene on the ancient-core ↔ MTBC-specific axis: a gene absent even from the closest MTBAP relatives is a candidate MTBC-specific innovation (possible host-adaptation factor, to confirm by synteny). The phylostratum extends that axis outside the genus (tblastn vs 13 reference genomes spanning Mycobacteriaceae → Corynebacteriales → Actinomycetia → outside the phylum): it is the deepest clade in which a homolog is still detected, i.e. a proxy for gene age. Read it with the null model in mind: a shallow (young) stratum can also reflect homology-detection failure for short or fast-evolving ORFs, so it is a descriptive axis, not a proof of novelty.

Essentiality (transposon mutagenesis)

Genome-wide Himar1 transposon essentiality in H37Rv (DeJesus 2017). An essential call (ES/ESD/GD) is strong, independent evidence that a "hypothetical" locus encodes a functional, selectively required gene — orthogonal to intra-species conservation.

Proteomics (mass spectrometry) detected

Detection by mass spectrometry is direct, experimental evidence that the protein product exists — orthogonal to sequence conservation and to Tn-seq essentiality, and especially decisive for a "hypothetical" locus. Read the dataset count as abundance, not as replication. Measured across this atlas (3,491 detected proteins), the number of datasets a protein appears in tracks its abundance almost monotonically: median 1.2 ppm at 1 dataset of 16, 27 ppm at 10, 392 ppm at 15, 1,325 ppm at 16 — three orders of magnitude. A high count therefore says the protein is abundant more than it says several independent experiments confirmed it. What the count does establish, at any value, is that the product exists.

Predicted localisation (DeepTMHMM + lipobox) no export signal, no TM helix

A verified absence of signals, which is not a compartment. No Sec/Tat signal peptide, no transmembrane helix and no lipobox were found on this sequence. The predictor reports topology and export signals; it does not measure where the protein ends up, so this page draws no cytoplasmic conclusion from it. In M. tuberculosis that reservation is not theoretical: the 12 Esx paralogues of this atlas — EsxA/ESAT-6 and EsxB/CFP-10 among them, the best-established secreted proteins of the species — carry this very call, because substrates of the type VII (ESX) secretion system leave no recognisable signal. « Secreted » and « no signal peptide » are true together here.

Transmembrane topology and signal peptide from DeepTMHMM (deep-learning reference predictor); lipoproteins from a (myco)bacterial lipobox motif. A sequence-based prediction of subcellular context.

Physico-chemical properties (computed, ProtParam)

Computed from the ancestral MTBC0 sequence with the ExPASy ProtParam method (Biopython). Descriptive biophysical context: a positive GRAVY flags a hydrophobic (often membrane) protein, a high instability index (>40) predicts a short in-vitro half-life, an extreme pI hints at compartment or binding partner.

Domains (Pfam, hmmscan --cut_ga)

3D structure (interactive — Mol*/pdbe-molstar)

No experimental structure is deposited for this gene: showing the AlphaFold DB predicted model (mean pLDDT 94.6, very high), coloured by per-residue confidence (dark blue = very high, orange/red = low — treat low-confidence regions as unreliable, especially termini and loops). Drag to rotate, scroll to zoom.

Structural search (AlphaFold DB model, Foldseek vs PDB — genome-wide) pLDDT 94.6

Foldseek search of the AlphaFold DB model (mean pLDDT 94.6, gated at 70) against the PDB — a genome-wide extension of the ESMFold dark-gene search that also covers proteins beyond the single-sequence length limit. Confident structural neighbours (E < 0.01) shown.

Genomic context (neighbours & predicted operon)

Neighbours from the H37Rv annotation (- strand). The operon is predicted by co-directional intergenic distance (same strand, gaps ≤50 bp) — a transcription-unit hypothesis, not a mapped TSS. For a "hypothetical", co-transcription with a characterised operon is a concrete functional lead (complements the STRING neighborhood channel below).

Published operon evidence (two independent sources)

Two independent published operon maps, cited alongside the distance heuristic above: an association corroborating or contradicting the heuristic, never proof of shared function. REMap's RNA-seq coverage continuity is the most direct of the three signals; Roback's logistic regression and the intergenic-distance heuristic are statistical proxies.

Node colour = verdict, dashed = still uncharacterised; edge colour = evidence (green experimental, orange genomic-context, grey co-expression), width ∝ score. Click a partner to open its page; "Explore full network" to walk the graph.

Closest characterised functional partner: ligC (DNA ligase C), high confidence (score 995, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence).

All 8 partner(s) at medium confidence are shown. None rests on an interaction measured in M. tuberculosis itself.

STRING combines evidence channels (neighborhood, fusion, cooccurrence, coexpression, experimental, database, text-mining) into a 0–1000 score. The ctx badge marks edges carried by the genomic-context channels (conserved neighborhood, fusion, phylogenetic co-occurrence), which are independent of orthology and structure and the strongest signal for an unknown gene. The no text-mining column recomputes the score from data alone, so a link that does not depend on the literature is visible. Association is a function hypothesis, not proof: corroborate with the operon context and the primary literature before assigning a function.

Where the experimental evidence comes from, and why it matters here. STRING's experimental channel fuses interactions measured in M. tuberculosis with interactions measured in another organism and transferred to this pair by orthology. The two are not equivalent for this atlas, whose standing rule is that better studied elsewhere is not a function established in M. tuberculosis. Measured on the served network: of the 38 229 partner pairs shown across the atlas, 13 483 partner rows carry a non-zero experimental channel, and only 106 pairs rest on an interaction measured in M. tuberculosis itself — 0.3 % of the network. Those 106 are long-established complexes (ESX-1/ESAT-6, RNA polymerase with CarD and RbpA, the Pup–Mpa proteasome, gyrase, Emb, GarA with PknB and PknG, the Rel modules); no still-uncharacterised gene is among them. The reason is not a gap in this atlas but in the literature: there is almost no experimental interactome of this bacterium, IntAct holding 134 curated protein pairs for a ~4 000-gene proteome. A exp (transferred) badge is therefore a lead, never a measurement, and an absence of badge is not evidence that the pair does not interact.

Evidence

• Legacy H37Rv annotation: hypothetical protein
• MTBC0 PGAP product: non-homologous end-joining DNA ligase
• Pfam (hmmscan --cut_ga): LigD_Prim-Pol PF21686.4 (E=5e-88), DNA_primase_S PF01896.26 (E=4e-10)
• (auto-curated by rules from PGAP + Pfam + Foldseek; not hand-reviewed)

Ancestral MTBC0 protein sequence

>mtbc0_003955|Rv3730c|ligD
MAAAAEELDVDGIAVRLTSPDRMYFPKLGSHGTKRRLVEYYFAVAGGPMLTALRDRPTHLQRFPDGVDGEQIYQKRIPRHRPDYLQTCRVTFPSGRMADALKVTHPAAIVWAAQMGTITLHPWQVRCPDTEHPDELRIDLDPQPGTGFVEARTVAVDVLRSVLDDLGLVGYPKTSGGRGIHVFLRIATDWDFVEVRRAGIALAREVERRAPDAVTTSWWKEERGARIFIDFNQNARDRTMASAYSVRPTPIATVSMPLTWEELAGADPDDYTMTTVPELVKIRDDPWAGMDDVAQSIAPLLDLAAADEERGLGDMPYPPNYPKMPGEPKRVQPSRDTDLKGGNTSK