PcrB is the heptaprenylglyceryl phosphate (HepGP) synthase of Bacillus subtilis, a homodimeric Mg2+-dependent TIM-barrel prenyltransferase of the GGGPS family (group I subfamily). It transfers the C35 heptaprenyl moiety of all-trans-heptaprenyl diphosphate to the C3 hydroxyl of sn-glycerol 1-phosphate, forming an ether bond and releasing diphosphate. This chemistry resembles archaeal ether-lipid synthesis despite being experimentally established in bacteria. The acceptor is the sn-1 glycerophosphate enantiomer used by archaeal membrane lipids, and the product carries an isoprenoid chain in ether rather than ester linkage. Published acceptor assays support sn-glycerol 1-phosphate specificity, while shorter C10 and C20 prenyl donors are accepted in vitro with much lower efficiency, and dimerization is what allows the long native donor to be bound. Unlike the archaeal pathway, the Bacillales product is not elaborated into a bulk membrane lipid by addition of a second prenyl chain. HepGP is instead dephosphorylated by a non-specific phosphatase and O-acetylated by YvoF, giving acetylated heptaprenylglycerol (Ac-HepG), a low-abundance lipophilic acetyl carrier. Ac-HepG supplies acetyl groups at the extracytoplasmic face of the membrane through an unresolved translocation route, where LhaT (YpjA) transfers them to the N-terminal cysteine of mature diacylglyceryl lipoproteins, and loss of pcrB abolishes that lipoprotein N-acetylation. PcrB is therefore the committed, ether-bond-forming step of a prenyl-lipid acetyl-carrier pathway, and has a broader distribution than LhaT; the lipoprotein-acetylation role cannot therefore be assumed for every PcrB homolog.
| GO Term | Evidence | Action | Reason |
|---|---|---|---|
| GO:0000287 magnesium ion binding | IEA GO_REF:0000104 | ACCEPT | Summary: Magnesium dependence supports cofactor binding as an integral part of PcrB catalysis. Reason: The full primary study reports no product after MgCl2 omission or EDTA addition, and inactive D14A mutants of both B. subtilis and S. aureus PcrB. Together with the curated Mg2+ cofactor statement, this supports the electronic binding claim. The residue-based coordination model is proposed rather than a native metal-occupancy measurement. A required catalytic cofactor is central to the characterized transferase function; it is not made peripheral simply because the reaction has a more informative activity term. Supporting Evidence: PMID:23322418 No product was found, indicating that Mg2+ is likely essential for catalysis. file:BACSU/pcrB/pcrB-uniprot.txt Name=Mg(2+); Xref=ChEBI:CHEBI:18420 |
| GO:0004659 prenyltransferase activity | IBA GO_REF:0000033 | MODIFY | Summary: True but less specific than the characterized reaction; refine to the HepGP synthase activity the gene already carries by IDA and EXP. Reason: The ancestral generic prenyltransferase claim agrees with direct characterization of B. subtilis PcrB, and target O34790 in WITH/FROM is expected experimental grounding rather than circularity. GO:0120536 specifies this target's established donor and acceptor and is the supported refinement. No lineage IRD/IKR was returned by the normal PAINT-loss check. This conclusion does not reconstruct an uninspected ancestral clade or grant C35 specificity to every group I enzyme from the short donor list. Propagation Review Root cause: TERM SCOPING PROBLEM Failure modes: GRANULARITY MISMATCH Sources checked: PANTHER:PTN002219408 SUPPORTS TRANSFER The exact IBD placement was not independently reconstructed. The generic prenyltransferase assertion is compatible with direct experimental evidence on the target; the normal lineage-loss check reports no IRD/IKR. Specificity is supplied by target assays, not inferred from donor counts. UniProtKB:O34790 SUPPORTS TRANSFER The target's own appearance among the sources is expected and not circular. PcrB has experimental annotations for the specific reaction, so it is one of the descendant evidences available when the ancestral assertion was placed, and its presence here marks experimental grounding on the target itself. UniProtKB:Q53726 SUPPORTS TRANSFER Listed descendant evidence for the ancestral assertion; a donor-specific assay/strain mapping was not independently reconstructed here. The target activity is directly established. UniProtKB:Q8Y6C8 SUPPORTS TRANSFER Listed descendant evidence; no inference of whole-clade C35 specificity is made from this donor label alone. Proposed replacements: heptaprenylglyceryl phosphate synthase activity Supporting Evidence: PMID:24684232 differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases file:BACSU/pcrB/pcrB-uniprot.txt Belongs to the GGGP/HepGP synthase family. Group I subfamily. |
| GO:0004659 prenyltransferase activity | IEA GO_REF:0000104 | MODIFY | Summary: Same claim as the IBA row, from the HAMAP rule for the family; correct but less specific than the experimentally established reaction. Reason: HAMAP-Rule MF_00112 covers both the GGGP and the HepGP synthases, so the rule can only assert the generic prenyltransferase activity. For this member the specific reaction has been measured directly, and UniProt itself records the specific EC number and reaction, so the specific descendant GO:0120536 is the right granularity. Consistent with the IBA row on the same term. Proposed replacements: heptaprenylglyceryl phosphate synthase activity Supporting Evidence: file:BACSU/pcrB/pcrB-uniprot.txt Reaction=sn-glycerol 1-phosphate + all-trans-heptaprenyl diphosphate = 3-heptaprenyl-sn-glycero-1-phosphate + diphosphate; PMID:21761520 PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. |
| GO:0006650 glycerophospholipid metabolic process | IEA GO_REF:0000041 | MODIFY | Summary: Correct, but the biosynthetic descendant the gene already carries is the supported granularity. Reason: The UniPathway mapping is faithful to the UniProt pathway statement, and the claim is true, since HepGP is a glycerophospholipid. PcrB only makes HepGP and does not degrade it, so the metabolic parent is less precise than the biosynthetic child GO:0046474, which the gene already carries by IDA. This is a refinement, not a correction. Proposed replacements: glycerophospholipid biosynthetic process Supporting Evidence: file:BACSU/pcrB/pcrB-uniprot.txt Membrane lipid metabolism; glycerophospholipid metabolism. PMID:21761520 PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. |
| GO:0016765 transferase activity, transferring alkyl or aryl (other than methyl) groups | IEA GO_REF:0000002 | MODIFY | Summary: The InterPro2GO mapping is sound for the GGGPS/HepGP signature but stops far above the characterized reaction. Reason: IPR008205 is the GGGP/HepGP synthase signature, which spans both the GGGP synthases and the HepGP synthases, so a transferase grandparent term is the most the family-level mapping can assert. The protein also matches the group I specific signature IPR039074 and HAMAP-Rule MF_00112, and its reaction has been measured, so GO:0120536, a descendant of this term and already asserted on the gene, is the supported granularity. Refinement, not correction. Proposed replacements: heptaprenylglyceryl phosphate synthase activity Supporting Evidence: PMID:24684232 differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases file:BACSU/pcrB/pcrB-uniprot.txt Belongs to the GGGP/HepGP synthase family. Group I subfamily. |
| GO:0046474 glycerophospholipid biosynthetic process | IBA GO_REF:0000033 | ACCEPT | Summary: The process PcrB is committed to; its immediate product, 3-heptaprenyl-sn-glycero-1-phosphate, is a glycerophospholipid, and no more specific GO term for this pathway exists. Reason: The node placement is sound. The clade is the group I GGGPS subfamily, whose members are HepGP synthases, and the appearance of O34790 among the sources is the expected marker that the node rests in part on experimental evidence on this gene. The claim itself is also directly established, so the row is not carrying the inference alone. Granularity was checked against the whole branch. No descendant of this term names heptaprenylglyceryl phosphate or any archaeal-type ether lipid, so this is as precise as GO currently allows, which is why it is accepted rather than modified. It is retained as core because making this lipid is the only process PcrB participates in. Propagation Review Root cause: NO FAILURE CORE Sources checked: PANTHER:PTN002219408 SUPPORTS TRANSFER Same node as the prenyltransferase IBA row, assessed the same way; no PAINT slice is cached locally. All donors are Bacillales group I HepGP synthases, so the inherited process claim is the synthesis of the heptaprenylglyceryl lipid, which is exactly what the target does. Unlike the molecular function row, the process term here is not refinable, since GO has no descendant for this lipid, so the propagated term is kept. UniProtKB:O34790 SUPPORTS TRANSFER The target among its own sources, as expected when the gene carries experimental evidence for the claim; this marks grounding on the target, not circularity. UniProtKB:Q53726 SUPPORTS TRANSFER Staphylococcus aureus PcrB (PCRB_STAA8), same subfamily. UniProtKB:Q8Y6C8 SUPPORTS TRANSFER Listeria monocytogenes PcrB (PCRB_LISMO), same subfamily. Supporting Evidence: PMID:21761520 PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. file:BACSU/pcrB/pcrB-uniprot.txt Membrane lipid metabolism; glycerophospholipid metabolism. |
| GO:0046474 glycerophospholipid biosynthetic process | IDA PMID:21761520 Functional assignment of an enzyme that catalyzes the synthe... | ACCEPT | Summary: Direct evidence that PcrB makes heptaprenylglyceryl phosphate in vitro and that the lipid and its derivatives are present in vivo and lost on deletion. Reason: This is the anchoring experimental row. Guldan et al. assayed the purified enzyme, identified HepGP as the product, and showed that cells lacking pcrB no longer make the dephosphorylated and acetylated derivatives of HepGP, so the gene product is responsible for building this glycerophospholipid in the cell rather than merely being capable of the reaction. The cached record is abstract-only, but the curator read the full text and the claim is independently recorded in the UniProt FUNCTION and DISRUPTION PHENOTYPE statements with the same reference. Core. Supporting Evidence: PMID:21761520 PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. file:BACSU/pcrB/pcrB-uniprot.txt Cells lacking this gene show a cloggy growth and do not produce the dephosphorylated and acetylated derivatives of HepGP. |
| GO:0046474 glycerophospholipid biosynthetic process | IEA GO_REF:0000120 | ACCEPT | Summary: Same claim as the IBA and IDA rows, from the HAMAP rule and an association rule; correct and consistent with them. Reason: One of the two sources is an ARBA association rule, which on its own would be an unvetted prediction, but the other is HAMAP-Rule MF_00112 for this family and the identical claim is established directly for this protein in the same review, so nothing rests on the rule. Accepted for consistency with the IDA row on the same term rather than as independent evidence. Supporting Evidence: PMID:21761520 PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. file:BACSU/pcrB/pcrB-uniprot.txt Reaction=sn-glycerol 1-phosphate + all-trans-heptaprenyl diphosphate = 3-heptaprenyl-sn-glycero-1-phosphate + diphosphate; |
| GO:0120536 heptaprenylglyceryl phosphate synthase activity | EXP PMID:24684232 A comprehensive analysis of the geranylgeranylglyceryl phosp... | ACCEPT | Summary: The core molecular function, measured as part of a systematic biochemical survey of the GGGPS family that separates the bacterial group I HepGP synthases from the group II GGGP synthases. Reason: Peterhoff et al. characterized seventeen representative family members side by side and found that bacterial group I enzymes, of which B. subtilis PcrB is the exemplar, are heptaprenylglyceryl phosphate synthases, distinct from the group II bacterial enzymes that have archaeal-type GGGP synthase activity. That comparison is what makes the chain-length assignment to the C35 donor specific rather than generic prenyltransferase chemistry. The cached record is abstract-only, but the abstract states the group I assignment, and UniProt cites this reference for CATALYTIC ACTIVITY with experimental evidence. Core. Supporting Evidence: PMID:24684232 differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases PMID:24684232 family enzymes catalyse the formation of an ether bond between glycerol-1-phosphate and polyprenyl diphosphates |
| GO:0120536 heptaprenylglyceryl phosphate synthase activity | IDA PMID:21761520 Functional assignment of an enzyme that catalyzes the synthe... | ACCEPT | Summary: Direct assay of the purified enzyme with its native substrates, with the acceptor and donor specificity established in the accompanying work. Reason: This is the annotation the whole review turns on, and it is well grounded. The purified protein transfers the C35 heptaprenyl group from heptaprenyl diphosphate to the C3 hydroxyl of sn-glycerol 1-phosphate, the acceptor is strictly the sn-1 enantiomer, crystal structures with bound glycerol 1-phosphate show how it is held, and the native chain length depends on the homodimer. In vitro use of the shorter C10 and C20 donors is substrate range rather than a second physiological reaction, since the C35 donor is the one the enzyme is built for and the C35 product is what accumulates in the cell, so no additional activity is annotated for it. Core. Supporting Evidence: file:BACSU/pcrB/pcrB-uniprot.txt Prenyltransferase that catalyzes in vivo the transfer of the heptaprenyl moiety of heptaprenyl pyrophosphate (HepPP; 35 carbon atoms) PMID:21214173 PcrB uses G1P (1) exclusively as an acceptor PMID:22614947 activity assays showed that monomerization limits the length of accepted polyprenyl pyrophosphates to three isoprene units, whereas the native PcrB substrate contains seven isoprene entities |
| GO:0120536 heptaprenylglyceryl phosphate synthase activity | IEA GO_REF:0000120 | ACCEPT | Summary: Same claim as the EXP and IDA rows, derived from the Rhea reaction cross-reference on the UniProt entry; correct. Reason: RHEA:33495 is the balanced reaction UniProt records for this protein with experimental evidence, and it is the reaction GO:0120536 is defined on, so the electronic mapping lands exactly on the measured activity. Consistent with the two experimental rows for the same term; accepted for consistency rather than as independent evidence. Supporting Evidence: file:BACSU/pcrB/pcrB-uniprot.txt Reaction=sn-glycerol 1-phosphate + all-trans-heptaprenyl diphosphate = 3-heptaprenyl-sn-glycero-1-phosphate + diphosphate; PMID:23322418 PcrB catalyzes the production of heptaprenylglyceryl phosphate from heptaprenyl diphosphate and glycerol-1-phosphate |
| GO:0008611 ether lipid biosynthetic process | IDA PMID:21761520 Functional assignment of an enzyme that catalyzes the synthe... | NEW | Summary: Retain the historical ether-lipid biosynthesis proposal: PcrB directly forms the ether linkage of HepGP. Reason: PcrB itself catalyzes ether-bond formation between glycerol phosphate and an isoprenoid chain, so it performs the biosynthetic work. GO:0008611 permits an ether linkage to a glycerol carbon (normally C-1), without excluding G1P stereochemistry; it is not an ancestor or descendant of the retained GO:0046474. The 2026-11-06 comparator notes reported about 5,192 annotations, sampled eukaryotic plasmalogen enzymes, and zero Archaea/Bacillota hits. The 2026-11-07 recheck distinguishes 5,192 exact-term from 5,432 descendant-inclusive hits and finds 17 exact bacterial rows, including experimentally annotated Myxococcus CarF; thus this is not a first prokaryotic use. Same-role archaeal GGGPS P0C882, O26652 and O29844 still carry GO:0046474, not GO:0008611. HAMAP MF_00112 explicitly describes ether formation but maps the process only to GO:0046474, explaining an important propagation route for that gap; O29844 also has other mapping support, and no claim about universal curator intent follows. CarF modifies a vinyl ether precursor and does not settle the G1P-isoprenoid convention. Retain the historical NEW on direct PcrB chemistry and the broad definition, while explicitly requesting clarification of this unrepresented GGGPS chemistry. This asserts neither bulk membrane assembly nor downstream protein modification. Supporting Evidence: PMID:24684232 family enzymes catalyse the formation of an ether bond between glycerol-1-phosphate and polyprenyl diphosphates PMID:21761520 PcrB, the bacterial homologue of the archaea-specific geranylgeranylglyceryl phosphate synthase, produces heptaprenylglyceryl phosphate in bacillales. |
| GO:0005737 cytoplasm | IC PMID:40626731 Lipoprotein N-terminal modification in Bacillus: a new parad... | NEW | Summary: Retain the historical cytoplasm inference for the soluble carrier-synthesizing enzyme. Reason: The 2025 primary study explicitly places PcrB/YvoF carrier synthesis in the cytosol, consistent with soluble PcrB biochemical preparations and available cytoplasmic substrates. This is a contextual inference, not a native localization assay. It does not depend on proving EgsA supplies the G1P pool, nor on the annotation pattern of other family members. Preserve the historical cytoplasm payload without claiming experimental subcompartment resolution. Supporting Evidence: PMID:40626731 an acetylated heptaprenylglyceryl (Ac-HepG) carrier synthesized by PcrB/YvoF in the cytosol that is required for lipoprotein acetylation PMID:40626731 which is subsequently loaded with acetyl units using acetyl-CoA (Ac-CoA) on the cytoplasmic side of the membrane |
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Download this section (compressed HTML)Q: What is the appropriate process boundary for enzymes that synthesize carrier backbones subsequently used in protein modification? PcrB directly synthesizes the lipid precursor; YvoF loads the acetyl group and LhaT transfers it to protein. No additional lipoprotein-process annotation is proposed from the loss phenotype alone. Carrier recycling in vivo also remains unresolved.
Suggested experts: GO Consortium biocurators, GO biological process ontology editors
Q: Should the ether-lipid biosynthesis ontology or guidance explicitly discuss isoprenoid ethers on sn-glycerol 1-phosphate? The current definition accommodates the chemistry, but sparse prokaryotic annotation patterns merit checking without assuming they represent either deliberate exclusion or curator oversight.
Suggested experts: GO biological process ontology editors, archaeal and bacterial lipid biochemists
Q: Does GO want a way to describe a lipophilic group-carrier cycle, in which a lipid is built, charged, discharged and reused? The heptaprenylglycerol pathway makes a carrier rather than a membrane component, and nothing in the lipid biosynthesis branch distinguishes the two, which is why the purpose of this pathway is invisible in the gene's annotations.
Suggested experts: GO Consortium biocurators, lipid metabolism curators
Q: Is the in vitro use of C10 and C20 prenyl donors by PcrB of any physiological consequence in Bacillales, or purely substrate range for an enzyme built around the C35 donor? The 2025 reconstitution used the shorter geranylgeranyl donor for convenience, so the shorter products are known to be usable by the downstream enzymes.
Suggested experts: Bacterial prenyltransferase biochemists
Experiment: Delete or conditionally deplete araM and measure heptaprenylglycerol, acetylated heptaprenylglycerol and acetylated lipoprotein by mass spectrometry, alongside chiral analysis of the glycerol-phosphate pool. Loss of the lipid would establish the flux from EgsA to PcrB; its persistence would show that a second, unidentified route to the sn-1 enantiomer operates in B. subtilis.
Hypothesis: EgsA (AraM) is the physiological source of the sn-glycerol 1-phosphate that PcrB uses.
Type: targeted deletion with lipidomics and chiral metabolite analysis
Experiment: Feed cells or proteoliposomes isotopically labelled heptaprenylglycerol and follow the label through the acetylated and free forms while lipoprotein acetylation proceeds, then compare the number of acetyl groups transferred per molecule of carrier. A ratio well above one would establish a catalytic cycle and place a regeneration step in the pathway.
Hypothesis: The ether lipid is a genuine carrier that is regenerated and reused, rather than a lipid consumed once per acetyl group delivered.
Type: isotope tracing with in vitro reconstitution
Experiment: In a Bacillus species that encodes PcrB but a pseudogenized LhaT, compare wild type with pcrB and yvoF deletions by untargeted lipidomics and by acetyl-proteomics of the extracytoplasmic and membrane fractions, and test the deletions for membrane-stress phenotypes. Any acetylated species or lipid lost with the carrier would identify the alternative use.
Hypothesis: PcrB is retained in strains lacking a functional LhaT because its product has another acceptor or another membrane role.
Type: comparative lipidomics and acetyl-proteomics in a non-model Bacillus
Experiment: Compare lipoprotein N-acetylation efficiency and carrier partitioning in reconstituted membranes using heptaprenyl (C35) and geranylgeranyl (C20) versions of the carrier, since the shorter form is known to be accepted by PcrB, YvoF and LhaT. A strong chain-length dependence in the transfer or flipping step would show that the C35 preference of PcrB is selected by the carrier cycle rather than by catalysis alone.
Hypothesis: The chain length of the prenyl donor matters for the carrier function, not just for the enzyme active site.
Type: in vitro reconstitution with defined carrier chain lengths
What is not known β curated, literature-grounded statements of the open unknowns (the inverse of core functions).
Gap: How the ether lipid PcrB makes reaches the outer face of the membrane, and whether the discharged carrier returns to the cytoplasmic face to be reloaded, is unknown. No transporter or flippase has been identified, and whether the movement is protein-mediated at all has not been determined.
OPEN BIOLOGY RESIDUAL_SUBGAP
What is known: What is established is that PcrB and YvoF build Ac-HepG on the cytoplasmic side from cytoplasmic substrates, that LhaT is an integral membrane protein acting on an acceptor on the outer leaflet, and that the whole pathway can be reconstituted in vitro from four purified activities with no transporter. What is unknown is whether a dedicated transporter exists, whether spontaneous flipping of the neutral lipid suffices, and whether HepG is recycled rather than discarded after the acetyl group is handed over.
Significance: Whether the pathway is a true carrier cycle or a one-way lipid flux decides how the PcrB reaction should be modelled, and in particular whether the product is consumed stoichiometrically or regenerated. It also determines whether a transport step belongs in the pathway at all.
What would resolve it: Identify the translocation step, by screening for mutants that accumulate cytoplasmic Ac-HepG while losing acetylated lipoprotein, and by measuring flipping of Ac-HepG and HepG in proteoliposomes with and without candidate transporters; then test recycling with isotopically labelled HepG.
Provenance (the field's own admissions):
Gap: Whether the sn-glycerol 1-phosphate that PcrB uses in the living cell is supplied by the glycerol-1-phosphate dehydrogenase EgsA (AraM) is not established, and whether any other route to that enantiomer operates in B. subtilis is unknown.
OPEN BIOLOGY BP_DARK
What is known: PcrB uses G1P and AraM can generate that enantiomer in vitro. The author dissertation includes ΞaraM and ΞpcrB lipid labeling after exogenous 14C-G1P feeding; this bypasses endogenous precursor synthesis and therefore does not establish native EgsA-to-PcrB flux. Un-fed deletion/complementation lipidomics resolving that connection was not identified.
Significance: Identifying the physiological precursor source distinguishes demonstrated PcrB chemistry from a complete native pathway assignment.
What would resolve it: Measure heptaprenylglycerol, its acetylated form and acetylated lipoprotein in an araM deletion and in a strain where araM is conditionally depleted, and quantify the glycerol 1-phosphate pool by chiral analysis; a strain lacking araM that still makes the lipid would point to an unidentified second route.
Provenance (the field's own admissions):
Gap: Whether the PcrB product has roles beyond supplying acetyl groups to lipoproteins is unknown. PcrB is present in Bacillales strains that lack a functional LhaT, and some species encoding both do not make acetylated lipoprotein, so the lipid may serve other extracellular acyl-transfer reactions or another function altogether.
OPEN BIOLOGY BP_DARK
What is known: What is established is that pcrB, yvoF and ypjA are each strictly required for lipoprotein N-acetylation in B. subtilis, and that the pathway reconstitutes in vitro. What is unknown is why PcrB is retained where the known consumer of its product is absent or pseudogenized, and whether any other acceptor of the carried acetyl group exists.
Significance: If Ac-HepG has other acceptors, PcrB participates in more than one process and the current single-pathway reading of the gene is incomplete; if it does not, the retention of pcrB without lhaT needs another explanation, such as a membrane-biophysical role for the neutral ether lipid.
What would resolve it: Compare the extracellular and membrane acetyl-proteome and lipidome of wild type with pcrB and yvoF deletions in a species that encodes PcrB but no functional LhaT, looking for acetylated macromolecules or lipid species lost with the carrier.
Provenance (the field's own admissions):
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