Rv3896c Resolved · medium

Annotation: from legacy to revised

Curation note: P16.23 (phase135, 2026-08-17): couche tss ajoutee (leaderless=False). P49.1 (phase233, 2026-09-15): couche tss re-clee (leaderless=False). 2026-09-18 (projet mtbc/Rv3896c-Rv3898c, P7.2): evidence/references enrichies (repli lysozyme-like confirme par Foldseek restreint au domaine 205-285, site actif E215/GVWQ aligne sur Slt70 et les Rpf), auto=false. Verdict et confidence INCHANGES : un seul appui structural, la barre des deux appuis independants de protocole_flip_dark.md n'est pas franchie (P4.8 encore ouverte).

In the literature (TB corpus sweep) named only in full text

No publication in PubMed mentions this gene in its title or abstract — not under its H37Rv locus tag, nor under any of its ortholog identifiers (M. bovis, M. marinum, M. smegmatis, M. leprae, M. abscessus). The atlas annotation rests on sequence/structure evidence, not on a primary study of this gene.

Scope of the sweep: What was actually swept: the H37Rv locus tag and the ortholog locus tags (M. bovis Mb…, M. marinum MMAR_…, M. smegmatis MSMEG_…, M. leprae ML…, M. abscessus MAB_…), plus the gene name where one exists. NOT swept: protein and enzyme names, including family shorthands such as Ldt(Mt4) or VapC28, and their orthographic variants. An absent locus-tag mention is therefore evidence about the TAG, and only indirect evidence about the protein (P18.14). Extended on 2026-08-10 (P18.20): the UniProt protein name and its spelling variants were swept as well; the result is in `protein_name_sweep`.

A verified absence of literature is itself information: it flags an annotation with no primary study behind it. 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 0.77 (95% CI -1.04 to 3.60). 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=0.230 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=6 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 (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) pseudogene candidate

Read the disruption percentage against its ceiling, not against 100%. The strain collection is deeply imbalanced by clade, so a loss fixed in an entire lineage still shows a small overall fraction: a gene disrupted in every L6 strain caps at 0.96% of the collection, in every M. caprae strain at 0.20%, in every M. microti strain at 0.02%. Of the 705 clades in this collection, 689 have a ceiling below 1%. A figure of "0.15% of strains" is therefore uninterpretable on its own — it can be noise or a complete, lineage-defining loss. Use the clade attribution row above, which gives the sub-clade sizes, and never read the percentage alone.

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) Mycobacterium

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) not detected

Not detected in the mass-spectrometry datasets integrated by PaxDb. This is not evidence of absence, and this atlas can say which bias dominates, because it measured it on its own 482 undetected proteins. Size is the main factor: undetected proteins have a median length of 145 aa against 295 aa for detected ones, and 25.3 % of them are shorter than 100 aa against 6.0 %. Predicted localisation matters far less than usually assumed: detection rates are 89.3 % for globular, 84.9 % for transmembrane, 88.0 % for lipoproteins and 77.8 % for signal-peptide proteins — so a short protein is under-sampled whatever its predicted compartment, and hydrophobicity alone is a weak excuse. Condition-specific expression and few tryptic peptides (PE/PPE repeats) add to this.

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)

No Pfam-A domain above the gathering threshold (hmmscan --cut_ga scan completed).

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

No experimental structure is deposited for this gene: showing the AlphaFold DB predicted model (mean pLDDT 80.7, confident), 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.

Genomic context (neighbours & predicted operon) operon of 3

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.

Transcriptional regulation (signed TRN: ChIP-seq + TFOE)

Regulatory edges from the ISB signed transcriptional regulatory network (TF ChIP-seq binding, Minch 2015 + TF-overexpression response, Rustad 2014). An edge is regulatory evidence (binding and/or expression change), not necessarily direct. For a "hypothetical", membership in a known regulon (e.g. DosR dormancy, PhoP virulence) is a physiological-context lead, not a function.

Condition: Condition-blind by construction. These edges come from transcription-factor OVEREXPRESSION in a single laboratory condition, so a listed regulator is not a claim that it governs this gene in every circumstance. Measured counter-examples exist within this atlas (see Rv2516c, where the DosR regulon collapses under moxifloxacin while the gene itself rises). Read a regulator as 'capable of shifting this gene', and look for the condition before building a hypothesis on it.

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: eccC2 (ESX-2 type VII secretion system protein EccC), high confidence (score 818, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence).

Showing the 15 strongest of this gene's 42 partners at medium confidence (4 at high confidence). The table is an extract, not a neighbourhood: across the proteome the real median degree is 34 while the served median is 15, so no hub, degree distribution or centrality may be read on this table. None of the 42 partners 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: transglycosylase
• (initial automatic assignment: auto-curated by rules from PGAP + Pfam + Foldseek; superseded by hand review)
• UniProt protein name 'Transglycosylase' (O05448) is machine-generated by ProtNLM (evidence ECO:0008006, source Google), annotationScore 1/5, proteinExistence 'Predicted' -- not independently validated by homology or experiment (project mtbc/Rv3896c-Rv3898c, P4.3, 2026-09-10).
• InterPro IPR023346 / SUPFAM SSF53955 (Lysozyme-like domain superfamily) over residues 210-279 of the 302-aa H37Rv allele; the grand-clade frameshift (codon 250) removes 43% of this domain (250-279), the structurally best-modelled region of the protein (AlphaFold pLDDT median 96.9 vs 80.6 for the 210-249 region retained) -- the catalytic core is destroyed, not a peripheral extension (project mtbc/Rv3896c-Rv3898c, P4.5, 2026-09-10/11).
• Foldseek restricted to the 205-285 domain (full-length protein gives zero significant hits, a dilution artefact) returns lytic-transglycosylase folds as best TM-score hits (0.81-0.87): Slt70 of E. coli (PDB 1QSA/1SLY) and M. tuberculosis Rpf domains (PDB 1XSF/3EO5/4EMN/4CGE/4OW1). Active-site check: E215 superposes on Slt70's catalytic Glu478 (Thunnissen et al. 1995 -- structural inference from a transition-state analogue, not mutagenesis) and on the Rpf catalytic glutamate (Cohen-Gonsaud et al. 2005, functional evidence; mutants E54K/E54A tested in vivo by Zhao et al. 2015); motif GVWQ (238-241) aligns with Slt70's GLMQ (493-496). E215 and GVWQ both lie before codon 250, so the grand-clade allele keeps an intact catalytic site inside a domain whose fold is otherwise destroyed (project mtbc/Rv3896c-Rv3898c, P4.5-P4.7, 2026-09-10/15).
• This is ONE structural appui, well corroborated but not yet independently confirmed by a second, unrelated line of evidence as protocole_flip_dark.md requires: STRING network partners (score>=700) tested and rejected as non-independent (dominated by genomic neighbourhood -- the same ESX-2 synteny fact already used above); a Boltz-2 peptidoglycan-docking campaign was in progress as of 2026-09-15. Verdict left unchanged pending that second appui (project mtbc/Rv3896c-Rv3898c, P4.8, still open).
• Second independent appui SOUGHT (2026-09-20, project mtbc/Rv3896c-Rv3898c P4.11) and NOT obtained: (i) a Boltz-2 peptidoglycan-docking campaign (GlcNAc-MurNAc vs the full 302-aa allele, positive control RpfE catalytic domain PDB 4CGE, negative control Rv3898c) returned a positive interface signal, but derives from the same Foldseek-based structural model as the appui above and does not count as independent (P4.8/P4.11); (ii) dN/dS of the grand-clade post-frameshift ORF tail (288-aa allele, codons 250-288) across 14,918 carriers (L5/L6/L9/L10/Bovis, `bdd/actuelle`): 9 independent low-frequency substitutions in the tail, 8 non-synonymous / 1 synonymous against a neutral expectation of N:S=2.55 (33.0 synonymous / 84.0 non-synonymous sites, Nei-Gojobori), observed pN/pS ~3.14 (above neutral -- no purifying selection signal, point estimate opposite to the functional hypothesis; exact binomial test p=0.46, not significant, underpowered at n=9). Both routes were the only ones not derived from the in-house 3D model; both are negative or inconclusive. The verdict below is therefore left unchanged, but now rests on a SINGLE non-independent structural appui, not the corroborated appui the wording previously implied -- protocole_flip_dark.md still requires a second, independent, convergent appui before this can be stated as more than a candidate fold resemblance (project mtbc/Rv3896c-Rv3898c, P4.11, P7.5, 2026-09-20).
• Genomic context corroboration, independent of homology/synteny: Das, Ghosh & Mande 2011 (PMC3227618) already identified the Rv3884c-Rv3896c island as a distinct secretion-system cluster (matching ESX-2) by sequence-composition anomaly detection, citing Rv3896c only as the island's boundary -- a genomic-context signal, not a function signal, and not usable as the second appui protocole_flip_dark.md requires (project mtbc/Rv3896c-Rv3898c, P4.1, P7.5, 2026-09-20).

Ancestral MTBC0 protein sequence

>mtbc0_004130|Rv3896c|
MSTWHRIGTEGEPLTDPLTTQAIAALSRGHGLFAGGVSGADIDAPQIQQYANAISWVANAVPTAAAYRWRGAARALRRLANTDEALAQIMAAAQIDHAHARTATRALLEAAKTDAMALTDTPLGRREAMARMAARLRAQHRHIARCRSRARLLGLRLRRLRYLRTAAARRPQVTTPGGRAQVLAAIQKALDIKGVHDPAARARWTRGMDLVARRESNYNANAINHWDSNAARGTPSRGVWQFIAPTFAAYHEPGTSTNIHDLVAQACAFINYARGHYGVAADASNLADLIQQADPRRSPRGY