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- PDB-8i6v: Cryo-EM structure of the polyphosphate polymerase VTC complex(Vtc... -

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Basic information

Entry
Database: PDB / ID: 8i6v
TitleCryo-EM structure of the polyphosphate polymerase VTC complex(Vtc4/Vtc3/Vtc1)
Components
  • (Vacuolar transporter chaperone complex subunit ...) x 2
  • Vacuolar transporter chaperone 3 complex subunit 3
KeywordsMEMBRANE PROTEIN / polyphosphate polymerase / VTC complex / coupled synthesis and translocation
Function / homology
Function and homology information


: / vacuolar transporter chaperone complex / ATP-polyphosphate phosphotransferase / polyphosphate biosynthetic process / engulfment of target by autophagosome / polyphosphate kinase activity / vacuole fusion, non-autophagic / microautophagy / polyphosphate metabolic process / vacuolar transport ...: / vacuolar transporter chaperone complex / ATP-polyphosphate phosphotransferase / polyphosphate biosynthetic process / engulfment of target by autophagosome / polyphosphate kinase activity / vacuole fusion, non-autophagic / microautophagy / polyphosphate metabolic process / vacuolar transport / inositol hexakisphosphate binding / fungal-type vacuole membrane / vacuolar membrane / autophagosome membrane / cell periphery / cell cortex / cytoplasmic vesicle / nuclear membrane / calmodulin binding / mRNA binding / endoplasmic reticulum membrane / endoplasmic reticulum / membrane
Similarity search - Function
VTC domain superfamily / Domain of unknown function DUF202 / VTC domain / Domain of unknown function (DUF202) / VTC domain / SPX domain / SPX domain profile.
Similarity search - Domain/homology
TRIPHOSPHATE / : / PHOSPHATE ION / Chem-POV / Vacuolar transporter chaperone complex subunit 1 / Vacuolar transporter chaperone complex subunit 4 / Vacuolar transporter chaperone 3 complex subunit 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsMayer, A. / Wu, S. / Ye, S.
Funding support China, 4items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0908500 China
Ministry of Science and Technology (MoST, China)2020YFA0908400 China
National Natural Science Foundation of China (NSFC)31971127 China
National Natural Science Foundation of China (NSFC)31900930 China
CitationJournal: EMBO J / Year: 2023
Title: Cryo-EM structure of the polyphosphate polymerase VTC reveals coupling of polymer synthesis to membrane transit.
Authors: Wei Liu / Jiening Wang / Véronique Comte-Miserez / Mengyu Zhang / Xuejing Yu / Qingfeng Chen / Henning Jacob Jessen / Andreas Mayer / Shan Wu / Sheng Ye /
Abstract: The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the ...The eukaryotic vacuolar transporter chaperone (VTC) complex acts as a polyphosphate (polyP) polymerase that synthesizes polyP from adenosine triphosphate (ATP) and translocates polyP across the vacuolar membrane to maintain an intracellular phosphate (P ) homeostasis. To discover how the VTC complex performs its function, we determined a cryo-electron microscopy structure of an endogenous VTC complex (Vtc4/Vtc3/Vtc1) purified from Saccharomyces cerevisiae at 3.1 Å resolution. The structure reveals a heteropentameric architecture of one Vtc4, one Vtc3, and three Vtc1 subunits. The transmembrane region forms a polyP-selective channel, likely adopting a resting state conformation, in which a latch-like, horizontal helix of Vtc4 limits the entrance. The catalytic Vtc4 central domain is located on top of the pseudo-symmetric polyP channel, creating a strongly electropositive pathway for nascent polyP that can couple synthesis to translocation. The SPX domain of the catalytic Vtc4 subunit positively regulates polyP synthesis by the VTC complex. The noncatalytic Vtc3 regulates VTC through a phosphorylatable loop. Our findings, along with the functional data, allow us to propose a mechanism of polyP channel gating and VTC complex activation.
History
DepositionJan 29, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Mar 1, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 5, 2023Group: Author supporting evidence / Structure summary
Category: pdbx_contact_author / pdbx_entity_instance_feature
Revision 1.2May 31, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Vacuolar transporter chaperone complex subunit 1
B: Vacuolar transporter chaperone complex subunit 1
C: Vacuolar transporter chaperone complex subunit 1
D: Vacuolar transporter chaperone 3 complex subunit 3
E: Vacuolar transporter chaperone complex subunit 4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)224,47511
Polymers223,1175
Non-polymers1,3586
Water724
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Vacuolar transporter chaperone complex subunit ... , 2 types, 4 molecules ABCE

#1: Protein Vacuolar transporter chaperone complex subunit 1 / Negative regulator of CDC42 protein 1 / Phosphate metabolism protein 4 / SPX-dependent ...Negative regulator of CDC42 protein 1 / Phosphate metabolism protein 4 / SPX-dependent polyphosphate polymerase VTC subunit 1 / Vacuolar membrane polyphosphate polymerase accessory subunit 1 / PolyP polymerase


Mass: 14388.053 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: VTC1, NRF1, PHM4, YER072W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40046
#3: Protein Vacuolar transporter chaperone complex subunit 4 / Phosphate metabolism protein 3 / Polyphosphate kinase / SPX-dependent polyphosphate polymerase VTC ...Phosphate metabolism protein 3 / Polyphosphate kinase / SPX-dependent polyphosphate polymerase VTC subunit 4 / Vacuolar membrane polyphosphate polymerase catalytic subunit / PolyP polymerase


Mass: 83268.664 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: VTC4, PHM3, YJL012C, J1345 / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P47075, ATP-polyphosphate phosphotransferase

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Protein , 1 types, 1 molecules D

#2: Protein Vacuolar transporter chaperone 3 complex subunit 3 / Phosphate metabolism protein 2 / SPX-dependent polyphosphate polymerase VTC subunit 3 / Vacuolar ...Phosphate metabolism protein 2 / SPX-dependent polyphosphate polymerase VTC subunit 3 / Vacuolar membrane polyphosphate polymerase accessory subunit 3 / PolyP polymerase


Mass: 96684.312 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: VTC3, PHM2, YPL019C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q02725

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Non-polymers , 5 types, 10 molecules

#4: Chemical ChemComp-POV / (2S)-3-(hexadecanoyloxy)-2-[(9Z)-octadec-9-enoyloxy]propyl 2-(trimethylammonio)ethyl phosphate / POPC / POPC


Mass: 760.076 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C42H82NO8P / Comment: phospholipid*YM
#5: Chemical ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: PO4
#6: Chemical ChemComp-3PO / TRIPHOSPHATE / Polyphosphate


Mass: 257.955 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: H5O10P3
#7: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Cryo-EM structure of the polyphosphate polymerase VTC complex(Vtc4/Vtc3/Vtc1)
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1042873 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00313456
ELECTRON MICROSCOPYf_angle_d0.47218191
ELECTRON MICROSCOPYf_dihedral_angle_d5.6811788
ELECTRON MICROSCOPYf_chiral_restr0.0382005
ELECTRON MICROSCOPYf_plane_restr0.0042298

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