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- EMDB-1618: 3D reconstruction of heterodimeric yeast Pol alpha using electron... -

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Basic information

Entry
Database: EMDB / ID: EMD-1618
Title3D reconstruction of heterodimeric yeast Pol alpha using electron microscopy
Map data3D reconstruction of heterodimeric yeast Pol alpha using electron microscopy
Sample
  • Sample: Yeast DNA polymerase alpha
  • Protein or peptide: DNA polymerase alpha
  • Protein or peptide: B subunit
KeywordsDNA replication / DNA Polymerase alpha / Electron microscopy
Function / homology
Function and homology information


protein binding / RNA-templated DNA biosynthetic process / nucleoside binding / premeiotic DNA replication / alpha DNA polymerase:primase complex / telomere capping / lagging strand elongation / DNA synthesis involved in DNA repair / DNA replication initiation / nuclear envelope ...protein binding / RNA-templated DNA biosynthetic process / nucleoside binding / premeiotic DNA replication / alpha DNA polymerase:primase complex / telomere capping / lagging strand elongation / DNA synthesis involved in DNA repair / DNA replication initiation / nuclear envelope / DNA-directed DNA polymerase activity / nucleotide binding / mitochondrion / DNA binding
Similarity search - Function
: / : / DNA polymerase alpha, subunit B, N-terminal / DNA polymerase alpha, subunit B / Zinc finger, DNA-directed DNA polymerase, family B, alpha / DNA polymerase alpha/delta/epsilon, subunit B / DNA-directed DNA polymerase, family B, multifunctional domain / DNA-directed DNA polymerase, family B, conserved site / DNA-directed DNA polymerase, family B, exonuclease domain / DNA-directed DNA polymerase, family B
Similarity search - Domain/homology
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 22.9 Å
AuthorsKlinge S / Nunez-Ramirez R / Llorca O / Pellegrini L
CitationJournal: EMBO J / Year: 2009
Title: 3D architecture of DNA Pol alpha reveals the functional core of multi-subunit replicative polymerases.
Authors: Sebastian Klinge / Rafael Núñez-Ramírez / Oscar Llorca / Luca Pellegrini /
Abstract: Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol alpha, Pol delta and Pol epsilon. The conserved catalytic and regulatory B subunits associate in ...Eukaryotic DNA replication requires the coordinated activity of the multi-subunit DNA polymerases: Pol alpha, Pol delta and Pol epsilon. The conserved catalytic and regulatory B subunits associate in a constitutive heterodimer that represents the functional core of all three replicative polymerases. Here, we combine X-ray crystallography and electron microscopy (EM) to describe subunit interaction and 3D architecture of heterodimeric yeast Pol alpha. The crystal structure of the C-terminal domain (CTD) of the catalytic subunit bound to the B subunit illustrates a conserved mechanism of accessory factor recruitment by replicative polymerases. The EM reconstructions of Pol alpha reveal a bilobal shape with separate catalytic and regulatory modules. Docking of the B-CTD complex in the EM reconstruction shows that the B subunit is tethered to the polymerase domain through a structured but flexible linker. Our combined findings provide a structural template for the common functional architecture of the three major replicative DNA polymerases.
History
DepositionMay 4, 2009-
Header (metadata) releaseMay 7, 2009-
Map releaseMay 6, 2011-
UpdateMay 6, 2011-
Current statusMay 6, 2011Processing site: PDBe / Status: Released

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Structure visualization

Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1618.tif

emd_1618.tif

Downloads & links

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Map

FileDownload / File: emd_1618.map.gz / Format: CCP4 / Size: 1001 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation3D reconstruction of heterodimeric yeast Pol alpha using electron microscopy
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.8 Å/pix.
x 64 pix.
= 243.2 Å		3.8 Å/pix.
x 64 pix.
= 243.2 Å		3.8 Å/pix.
x 64 pix.
= 243.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.8 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.045
Minimum - Maximum-0.0982105 - 0.13849
Average (Standard dev.)0.00211433 (±0.0123166)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 243.2 Å
α=β=γ: 90 °

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Supplemental data

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Sample components

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Entire : Yeast DNA polymerase alpha

EntireName: Yeast DNA polymerase alpha
Components
  • Sample: Yeast DNA polymerase alpha
  • Protein or peptide: DNA polymerase alpha
  • Protein or peptide: B subunit

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Supramolecule #1000: Yeast DNA polymerase alpha

SupramoleculeName: Yeast DNA polymerase alpha / type: sample / ID: 1000
Oligomeric state: One monomer of DNA polymerase alpha binds to a monomer of B-subunit
Number unique components: 2
Molecular weightTheoretical: 185 KDa / Method: Gel Filtration

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Macromolecule #1: DNA polymerase alpha

MacromoleculeName: DNA polymerase alpha / type: protein_or_peptide / ID: 1 / Name.synonym: DNA polymerase alpha / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 130 KDa / Theoretical: 130 KDa
SequenceGO: alpha DNA polymerase:primase complex, mitochondrion, DNA binding, DNA-directed DNA polymerase activity, nucleoside binding, nucleotide binding, protein binding, DNA replication initiation, DNA ...GO: alpha DNA polymerase:primase complex, mitochondrion, DNA binding, DNA-directed DNA polymerase activity, nucleoside binding, nucleotide binding, protein binding, DNA replication initiation, DNA synthesis involved in DNA repair, lagging strand elongation, premeiotic DNA replication, RNA-templated DNA biosynthetic process
InterPro: DNA-directed DNA polymerase, family B, DNA-directed DNA polymerase, family B, multifunctional domain, INTERPRO: IPR017966, DNA-directed DNA polymerase, family B, conserved site, DNA- ...InterPro: DNA-directed DNA polymerase, family B, DNA-directed DNA polymerase, family B, multifunctional domain, INTERPRO: IPR017966, DNA-directed DNA polymerase, family B, conserved site, DNA-directed DNA polymerase, family B, exonuclease domain, INTERPRO: IPR004578, Zinc finger, DNA-directed DNA polymerase, family B, alpha

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Macromolecule #2: B subunit

MacromoleculeName: B subunit / type: protein_or_peptide / ID: 2 / Name.synonym: B subunit / Number of copies: 1 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Molecular weightExperimental: 55 KDa / Theoretical: 55 KDa
SequenceGO: alpha DNA polymerase:primase complex, nuclear envelope, DNA binding, DNA-directed DNA polymerase activity, protein binding, DNA replication initiation, lagging strand elongation, telomere capping
InterPro: DNA polymerase alpha/delta/epsilon, subunit B, DNA polymerase alpha, subunit B, DNA polymerase alpha, subunit B, N-terminal

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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

Concentration0.7 mg/mL
BufferpH: 8 / Details: 100mM TrisHCl pH 8.0, 500 mM NaCl
StainingType: NEGATIVE
Details: A diluted solution of Pol alpha complex was adsorbed onto glow-discharged carbon coated grids, stained with 2% uranyl formate
GridDetails: 400 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1230
TemperatureMin: 293 K / Max: 293 K / Average: 293 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
DetailsMicroscope used JEOL-1230
DateAug 20, 2009
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 10 µm / Number real images: 170 / Average electron dose: 15 e/Å2 / Bits/pixel: 8
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.9 mm / Nominal defocus max: 1.2 µm / Nominal defocus min: 0.75 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Eucentric / Specimen holder model: JEOL

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Image processing

CTF correctionDetails: reverse phases
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 22.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, Xmipp / Number images used: 12913
Final two d classificationNumber classes: 190

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Atomic model buiding 1

Initial modelPDB ID:

3flo

SoftwareName: adpem
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation

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Atomic model buiding 2

Initial modelPDB ID:

2vwj

SoftwareName: adpem
DetailsProtocol: Rigid Body
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation

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