Rv3527 Family assigned · low

Annotation: from legacy to revised

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).

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.

Intrinsic disorder (sequence + structure) partially disordered

carries a substantial disordered region (53/149 residues); disorder is a property, not a function

A property (biophysics), not a function. No LLPS/condensate claim is made from disorder alone. Verdict unchanged. Source: metapredict v3 (Emenecker/Holehouse) per-residue disorder + AlphaFold mean pLDDT (annotation_mtbc P16.13).

Operon-context functional lead (hypothesis)

Co-transcribed within the operon kshA-Rv3527, pointing to cholesterol catabolism.

Basis: co-transcription in operon kshA-Rv3527 + purifying selection. This is a contextual candidate association (guilt-by-co-transcription), not an established molecular function.

Roback 2007 check: corroborated by the independently published operon map (Roback et al. 2007) — called group for this locus: Rv3526, Rv3527, predicted co-transcription probability for the cited neighbour: 0.651402. Roback et al. 2007 (peer-reviewed operon map, PMID 17652327) corroborates the co-transcription claimed above (cited operon: kshA-Rv3527; Roback's called group for this locus: Rv3526,Rv3527; predicted co-transcription probability for the cited neighbour: 0.651402). Verdict and function_revised are unchanged: the genomic neighbourhood and the named neighbour remain factual; only the co-transcription argument itself is now qualified by this independent source.

Two-source operon check: PARTAGÉ 1/2 Whether the CITED NEIGHBOUR Rv3526 is co-transcribed, according to each independently published map — Roback 2007: same operon; REMap 2016 (RNA-seq coverage): different operons. Base rates over the adjacent gene pairs that could be an operon at all, i.e. the co-directional ones: Roback 39.2 %, REMap 67.7 % — an agreement is only informative above its own base rate, and that rate has to be read on the right denominator (over ALL adjacent pairs, opposite-strand ones included, the same rates read 26.3 % and 45.6 %, diluted by pairs that cannot be co-transcribed). Three-way operon check (PARTAGÉ 1/2). The requalification argues co-transcription with Rv3526 (cited operon: kshA-Rv3527). Independent published maps: Roback et al. 2007 places it in the same operon (predicted probability 0.651402); REMap 2016 (RNA-seq coverage) does NOT place it in the same operon. This tests the claim actually made — whether the CITED NEIGHBOUR is co-transcribed — and not whether the three maps draw identical operon boundaries, which is a different question (strict group identity: Roback True, REMap False). Read each map against its own base rate, and against the RIGHT one: all twelve operon-based requalifications concern CO-DIRECTIONAL adjacent pairs, so the null is the rate over those pairs only — Roback calls co-transcription for 39.2 % of them (2429 pairs), REMap for 67.7 % (2230 pairs). Over all adjacent pairs, opposite-strand ones included, the same rates read 26.3 % and 45.6 %, but a divergent or convergent pair cannot form a transcription unit at all and neither map calls one (Roback 0.2 %, REMap 0.0 %), so that denominator dilutes the null instead of describing it. Against the conditioned null, REMap does not agree more often than chance across the twelve requalifications (9/10, one-sided binomial p = 0.116) and Roback does not either (5/12, p = 0.538). Correction of record (2026-10-09): until that date this sentence quoted the unconditioned null and reported REMap as significant (p = 0.00502); that significance does not survive the right denominator. So a lone REMap agreement is not a validation, and a lone Roback disagreement is weak evidence against the claim. Verdict, confidence and function_revised are unchanged; co-transcription remains an association, never a proof of shared function.

Conditional expression context (iModulons)

Member of 1 independently-modulated gene set(s): Rv0681 (Rv0681).

iModulon membership (independently-modulated gene sets from a 647-sample RNA-seq compendium): the conditional co-expression context. Co-expression is a regulatory context, NOT a molecular function. Source: iModulonDB / modulome_mtb (Yoo 2022).

CRISPRi vulnerability

Vulnerability index 1.04 (95% CI -0.49 to 3.43). 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=0.237 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=7 is descriptive only and does not predict this. Do not cite this VI as a standalone vulnerability claim.

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)

As checked on 2026-09-23, UniProt listed this protein as Uncharacterized protein; the revised annotation above goes beyond that record. UniProt may have been recurated since: check the live entry.

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) Corynebacteriales

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)

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 81.8, 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 2

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: kshA (3-ketosteroid-9-alpha-monooxygenase oxygenase subunit), high confidence (score 977, all STRING channels except text-mining; the partner is chosen on its genomic-context evidence).

Showing the 15 strongest of this gene's 79 partners at medium confidence (8 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 79 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
• Immediate genomic neighbour of kshA (Rv3526) on H37Rv; within the KstR1 cholesterol-catabolism regulon region (Kendall 2007)
• STRING context-driven association (text-mining excluded) to the cholesterol core (kshA, hsaC, hsaD, chsH1/2, kstR)
• Cholesterol module permutation test: 41/55 context edges, enrichment ~195x, z~84, p<5e-5 (cross-checked vs STRING ppi)
• Anchor KshAB is an essential Rieske [2Fe-2S] 3-ketosteroid 9alpha-hydroxylase required for pathogenesis (Capyk 2009; Hu 2010)
• Under purifying selection on 145,209 MTBC genomes (6 segregating sites)
• STRUCTURAL REFINEMENT (ESM Atlas, Lin 2023): 11/25 nearest ESM neighbours annotated as ferredoxins
• Fe-S motif: Cys78-x-x-Cys81 + distal Cys38/Cys107/Cys126 (iron-sulfur cluster coordination)
• Mechanistic fit: a Rieske oxygenase (KshA) requires an electron-transfer partner -> candidate ferredoxin electron donor of the KshAB cholesterol monooxygenase
• No dedicated functional study of Rv3527 in the PubMed TB corpus (tbmonitor, 2026-06)
• Guilt-by-association + structural-homology hypothesis; molecular role experimentally undemonstrated (verdict kept 'dark', auto=false). Decisive test: AlphaFold-multimer co-folding with KshA/KshB
• Curated by project conserved_orphan_modules (cholesterol module + structural refinement, 2026-06)

Ancestral MTBC0 protein sequence

>mtbc0_003743|Rv3527|
MPDDQPAVPDVDRLARSMLLLHGDHHDHNDSPEQHRTCGSWSKSRDFADDPQRAAAVREASRAERDRYLTSGLQPVDCRFCHVTVTVKRLGPGHTAVQWNTEASRRCAYFTELRARGGDSARTRSCPRLTDSIEHAVAEGYLEHHDPNR