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- EMDB-2409: The architecture of yeast DNA polymerase 'zeta'. Electron microsc... -

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Basic information

Entry
Database: EMDB / ID: EMD-2409
TitleThe architecture of yeast DNA polymerase 'zeta'. Electron microscopy reconstruction of the heterotetrameric complex Rev3-Rev7-Pol31-Pol32.
Map dataMap of Pol-zeta-d heterotetramer by negative stain
Sample
  • Sample: Polymerase Zeta heterotetramer of Saccharomyces cerevisiae
  • Protein or peptide: DNA Polymerase 'Zeta' processivity subunit
  • Protein or peptide: DNA Polymerase 'Delta' small subunit
  • Protein or peptide: DNA Polymerase 'Delta' subunit 3
  • Protein or peptide: DNA Polymerase 'Zeta' catalytic subunit
KeywordsTranslesion DNA synthesis / DNA repair / DNA replication / DNA polymerase zeta / genome integrity / cancer
Function / homology
Function and homology information


Translesion synthesis by REV1 / delta DNA polymerase complex / DNA amplification / zeta DNA polymerase complex / RNA-templated DNA biosynthetic process / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA replication, removal of RNA primer / lagging strand elongation / double-strand break repair via break-induced replication ...Translesion synthesis by REV1 / delta DNA polymerase complex / DNA amplification / zeta DNA polymerase complex / RNA-templated DNA biosynthetic process / Processive synthesis on the lagging strand / Removal of the Flap Intermediate / DNA replication, removal of RNA primer / lagging strand elongation / double-strand break repair via break-induced replication / postreplication repair / DNA strand elongation involved in DNA replication / DNA metabolic process / leading strand elongation / error-free translesion synthesis / mismatch repair / error-prone translesion synthesis / nucleotide-excision repair / double-strand break repair via homologous recombination / base-excision repair / 4 iron, 4 sulfur cluster binding / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / chromatin / mitochondrion / DNA binding / metal ion binding / nucleus / cytosol
Similarity search - Function
DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase zeta catalytic subunit / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / DNA polymerase delta/II small subunit family / DNA polymerase delta/II small subunit family / Mad2-like ...DNA polymerase delta subunit, OB-fold domain / DNA polymerase delta subunit 2, C-terminal domain / DNA polymerase delta subunit OB-fold domain / DNA polymerase zeta catalytic subunit / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / C4-type zinc-finger of DNA polymerase delta / DNA polymerase delta/II small subunit family / DNA polymerase delta/II small subunit family / Mad2-like / HORMA domain / HORMA domain / HORMA domain / HORMA domain profile. / HORMA domain superfamily / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/delta/epsilon, subunit B / DNA polymerase alpha/epsilon subunit B / DNA polymerase family B, thumb domain / DNA polymerase family B signature. / DNA-directed DNA polymerase, family B, conserved site / DNA-directed DNA polymerase, family B, conserved site / DNA polymerase family B / DNA polymerase family B, exonuclease domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B, multifunctional domain / DNA polymerase, palm domain superfamily / DNA polymerase, palm domain superfamily / DNA polymerase type-B family / DNA-directed DNA polymerase, family B / DNA-directed DNA polymerase, family B / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
DNA polymerase zeta catalytic subunit / DNA polymerase zeta processivity subunit / DNA polymerase delta small subunit / DNA polymerase delta subunit 3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 23.0 Å
AuthorsGomez-Llorente Y / Malik R / Jain R / Roy Choudhury J / Johnson RE / Prakash L / Prakash S / Ubarretxena-Belandia I / Aggarwal AK
CitationJournal: Cell Rep / Year: 2013
Title: The architecture of yeast DNA polymerase ζ.
Authors: Yacob Gómez-Llorente / Radhika Malik / Rinku Jain / Jayati Roy Choudhury / Robert E Johnson / Louise Prakash / Satya Prakash / Iban Ubarretxena-Belandia / Aneel K Aggarwal /
Abstract: DNA polymerase ζ (Polζ) is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall ...DNA polymerase ζ (Polζ) is specialized for the extension step of translesion DNA synthesis (TLS). Despite its central role in maintaining genome integrity, little is known about its overall architecture. Initially identified as a heterodimer of the catalytic subunit Rev3 and the accessory subunit Rev7, yeast Polζ has recently been shown to form a stable four-subunit enzyme (Polζ-d) upon the incorporation of Pol31 and Pol32, the accessory subunits of yeast Polδ. To understand the 3D architecture and assembly of Polζ and Polζ-d, we employed electron microscopy. We show here how the catalytic and accessory subunits of Polζ and Polζ-d are organized relative to each other. In particular, we show that Polζ-d has a bilobal architecture resembling the replicative polymerases and that Pol32 lies in proximity to Rev7. Collectively, our study provides views of Polζ and Polζ-d and a structural framework for understanding their roles in DNA damage bypass.
History
DepositionJul 2, 2013-
Header (metadata) releaseJul 31, 2013-
Map releaseOct 30, 2013-
UpdateOct 22, 2014-
Current statusOct 22, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0938
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0938
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2409.jpg

Downloads & links

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Map

FileDownload / File: emd_2409.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of Pol-zeta-d heterotetramer by negative stain
Voxel sizeX=Y=Z: 3.78 Å
Density
Contour LevelBy AUTHOR: 0.0938 / Movie #1: 0.0938
Minimum - Maximum-0.07746604 - 0.19041532
Average (Standard dev.)0.00239383 (±0.02202785)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions808080
Spacing808080
CellA=B=C: 302.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.783.783.78
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z302.400302.400302.400
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS808080
D min/max/mean-0.0770.1900.002

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Supplemental data

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Sample components

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Entire : Polymerase Zeta heterotetramer of Saccharomyces cerevisiae

EntireName: Polymerase Zeta heterotetramer of Saccharomyces cerevisiae
Components
  • Sample: Polymerase Zeta heterotetramer of Saccharomyces cerevisiae
  • Protein or peptide: DNA Polymerase 'Zeta' processivity subunit
  • Protein or peptide: DNA Polymerase 'Delta' small subunit
  • Protein or peptide: DNA Polymerase 'Delta' subunit 3
  • Protein or peptide: DNA Polymerase 'Zeta' catalytic subunit

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Supramolecule #1000: Polymerase Zeta heterotetramer of Saccharomyces cerevisiae

SupramoleculeName: Polymerase Zeta heterotetramer of Saccharomyces cerevisiae
type: sample / ID: 1000 / Details: Rev3:Rev7:Pol31:Pol32N 1:1:1:1 / Oligomeric state: Heterotetramer / Number unique components: 4
Molecular weightExperimental: 300 KDa / Theoretical: 298 KDa

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Macromolecule #1: DNA Polymerase 'Zeta' processivity subunit

MacromoleculeName: DNA Polymerase 'Zeta' processivity subunit / type: protein_or_peptide / ID: 1 / Name.synonym: Rev7 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Cytoplasm
Molecular weightExperimental: 28 KDa / Theoretical: 28 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: PETDuet
SequenceUniProtKB: DNA polymerase zeta processivity subunit
GO: error-free translesion synthesis, error-prone translesion synthesis
InterPro: HORMA domain

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Macromolecule #2: DNA Polymerase 'Delta' small subunit

MacromoleculeName: DNA Polymerase 'Delta' small subunit / type: protein_or_peptide / ID: 2 / Name.synonym: Pol31 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Cytoplasm
Molecular weightExperimental: 55 KDa / Theoretical: 55 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: PETDuet
SequenceUniProtKB: DNA polymerase delta small subunit
GO: DNA replication, removal of RNA primer, mismatch repair, base-excision repair
InterPro: DNA polymerase alpha/delta/epsilon, subunit B, DNA polymerase delta/II small subunit family

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Macromolecule #3: DNA Polymerase 'Delta' subunit 3

MacromoleculeName: DNA Polymerase 'Delta' subunit 3 / type: protein_or_peptide / ID: 3 / Name.synonym: Pol32
Details: We are using the N-terminal part of this protein. For further details, check the associated publication.
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Cytoplasm
Molecular weightExperimental: 40 KDa / Theoretical: 40 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: PETDuet
SequenceUniProtKB: DNA polymerase delta subunit 3
GO: DNA replication, removal of RNA primer, mismatch repair, base-excision repair

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Macromolecule #4: DNA Polymerase 'Zeta' catalytic subunit

MacromoleculeName: DNA Polymerase 'Zeta' catalytic subunit / type: protein_or_peptide / ID: 4 / Name.synonym: Rev3 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: Cytoplasm
Molecular weightExperimental: 173 KDa / Theoretical: 173 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: PETDuet
SequenceUniProtKB: DNA polymerase zeta catalytic subunit
GO: error-free translesion synthesis, error-prone translesion synthesis, DNA replication
InterPro: DNA-directed DNA polymerase, family B, DNA-directed DNA polymerase, family B, conserved site, DNA-directed DNA polymerase, family B, exonuclease domain, DNA-directed DNA polymerase, family ...InterPro: DNA-directed DNA polymerase, family B, DNA-directed DNA polymerase, family B, conserved site, DNA-directed DNA polymerase, family B, exonuclease domain, DNA-directed DNA polymerase, family B, multifunctional domain, DNA polymerase, palm domain superfamily, Ribonuclease H-like superfamily, C4-type zinc-finger of DNA polymerase delta

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8 / Details: 25mM Tris pH 8.0, 250mM NaCl
StainingType: NEGATIVE
Details: 2 ul aliquots were adsorbed onto glow-discharged carbon coated copper grids, and negatively stained with 2% uranyl acetate.
GridDetails: Glow discharged carbon coated copper grids.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 2100F
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 63450 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 40000
Sample stageSpecimen holder model: JEOL
TemperatureMin: 80 K / Max: 298 K / Average: 293 K
DateJan 1, 2011
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS / Average electron dose: 10 e/Å2
Tilt angle min0
Tilt angle max0

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Image processing

CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 23.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Xmipp, EMAN2, Spider / Number images used: 14844

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