Rv3224A Family assigned · low auto-curated · to review

H37Rv Rv3224A · MTBC0 · 62 aa · 3600635–3600823 H37Rv (+) · RefSeq

Genomic neighbourhood (genome browser)

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Annotation: from legacy to revised

Legacy (H37Rv / Mycobrowser)Conserved hypothetical protein
MTBC0 PGAP re-annotation
Revised (this work)Belongs to the prolyl-tRNA editing family. YbaK EbsC subfamily
Functional category (TubercuList)conserved hypotheticals

Curation note: Added P16.5d: Mycobrowser-annotated gene absent from RefSeq NC_000962.3 (the atlas' original 3906-CDS reference). Foundation record from the Mycobrowser release + translated H37Rv sequence; heavy enrichment layers (structure, orthology, conservation, interaction) pending.

Auto-curated: this verdict and function were generated by rules from PGAP + Pfam + Foldseek and have not been hand-reviewed (flagged for human review).

Legacy record & comparison (Mycobrowser)

Mycobrowser functionFunction unknown

The legacy Mycobrowser record is shown for verification. Mycobrowser is no longer maintained; its EC numbers predate recent nomenclature revisions, so a class change usually reflects re-numbering, not a conflict.

Curated reference (UniProt)

UniProt Q6MWZ5 TrEMBL · unreviewed
UniProt nameUncharacterized protein

UniProt still lists this protein as Uncharacterized protein; the revised annotation above is ahead of the current UniProt record.

Functional vocabulary (eggNOG-mapper, orthology transfer)

COG category S Function unknown
Preferred nameybaK
eggNOG descriptionBelongs to the prolyl-tRNA editing family. YbaK EbsC subfamily
Orthologous groupCOG2606
KEGG orthology ko:K03976

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, 145 209 strains)

pN/pS 0.386 · purifying
Polymorphic sites (≥ 0.1% of strains) 2 synonymous, 2 missense, 0 nonsense, 0 frameshift

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.

Essentiality (transposon mutagenesis)

DeJesus 2017 callNE · non-essential
What the call meansnon-essential
TA sites (Himar1) 4 in the ORF — 0 in the essential state, 0 growth-defect, 4 non-essential, 0 growth-advantage. Saturation 1.000, mean read count 84.25. A region of the protein devoid of TA sites is invisible to this assay: nothing can be inferred about it, in either direction.
CaveatStatistically thin call: only 4 TA (Himar1) sites in the whole ORF (atlas median 13; genes under 300 nt typically have very few). A DeJesus 2017 call built on so few independent observations is less robust than the same call on a longer gene, in either direction. Cross-check against the CRISPRi vulnerability index (independent of TA-site density) and, if this gene overlaps a neighbour (see Genomic-neighbour overlap section below), verify how many of its TA sites actually fall inside its own ORF. (P20.3)

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

MS detectiondetected in 1 of 16 independent MS datasets
Integrated abundance16.8 ppm · rank 2434/3519 (30.9th percentile)

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. Reproducible detection across several independent datasets (PaxDb) makes the existence claim robust; the integrated abundance places the protein in the proteome's dynamic range.

Physico-chemical properties (computed, ProtParam)

Length62 aa
Molecular weight6.5 kDa
Theoretical pI11.31
GRAVY-0.531 (hydrophilic)
Aliphatic index72.4
Aromaticity0.016
Instability index63.8 (unstable)

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 (or not yet scanned).

Functional interaction network (STRING v12, guilt-by-association)

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Node colour = verdict, dashed = hypothetical; 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: Rv3224 (iron-regulated short-chain dehydrogenase/reductase), high confidence from genomic context alone (score 849 excluding text-mining). This association is the citable seed of a function hypothesis for this hypothetical protein.

PartnerProductScoreNo text-miningChannels (≥400)
Rv3224 iron-regulated short-chain dehydrogenase/reductase 849 849 ctx neighborhood:781
Rv3224B Rv3224B, len: 72 aa. Conserved hypothetical protein (possibly gene fragment), similar to C-terminal part of ML0799|AL583919_131 conserved hy 808 808 ctx neighborhood:781
Rv3221A rshA anti-sigma factor RshA 420 420 ctx neighborhood:412
Rv3222c hyp hypothetical protein 418 418 ctx neighborhood:412
Rv3223c sigH ECF RNA polymerase sigma factor SigH 415 415 ctx neighborhood:412

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 exp badge marks an experimentally-supported partner (measured interaction, experimental/database channel ≥400) as opposed to a purely predicted one — but note that the M. tuberculosis experimental interactome is dominated by a noisy bacterial-two-hybrid screen, so a strong measured link that contradicts the operon/localisation context is likely a false positive. 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.

Sources

  • Ancestral sequence & coordinates: Harrison LB et al. (2024), An imputed ancestral reference genome for the MTBC, doi:10.1101/2023.09.07.556366
  • Product annotation: NCBI PGAP on MTBC0; legacy from H37Rv NC_000962.3 (RefSeq )
  • Domains: Pfam-A via hmmscan --cut_ga — none above threshold
  • Sequence-level signal: ESM Atlas (EvolutionaryScale × BioHub) — exploratory
  • Controlled vocabulary: eggNOG-mapper 2.1.12 (Cantalapiedra et al. 2021, doi:10.1093/molbev/msab293), eggNOG 5.0 DB (Huerta-Cepas et al. 2019) — OG COG2606
  • Curated reference: UniProt Q6MWZ5 (TrEMBL, unreviewed)
  • Intra-MTBC selection: pN/pS and disruption from SPDI variants of 145 209 MTBC strains (this work, local collection vs H37Rv NC_000962.3)
  • Interaction network: STRING v12.0 (Szklarczyk et al. 2023, doi:10.1093/nar/gkac1000), taxon 83332, CC-BY 4.0 — 5 functional partner(s); context anchor Rv3224
  • Essentiality: genome-wide transposon mutagenesis in H37Rv — DeJesus et al. 2017 (mBio, doi:10.1128/mBio.02133-16, CC BY)
  • Proteomics: integrated mass-spectrometry abundance from PaxDb 5.0 (Huang et al. 2023, doi:10.1016/j.mcpro.2023.100640), taxon 83332 — weighted average of 16 datasets, incl. Schubert et al. 2013 (doi:10.1016/j.chom.2013.04.008) and Albrethsen et al. 2013 (doi:10.1074/mcp.M112.018846)
  • Functional category: TubercuList scheme (Cole et al. 1998, doi:10.1038/31159), via Mycobrowser (Kapopoulou et al. 2011, doi:10.1016/j.tube.2010.09.006)
  • Physico-chemical properties: ExPASy ProtParam method via Biopython (Gasteiger et al. 2005), computed from the MTBC0 sequence
  • Primary literature: none located yet; annotation rests on the domain/homology sources above.

Ancestral MTBC0 protein sequence

>|Rv3224A|Rv3224A
VRRSASTCGWKTPTRRGTSRPSDSKTLILELPDERAVAIVPVPSKLSLKAAGGPRGAQSGHG