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- EMDB-5220: The architecture of the DNA polymerase-PCNA-DNA ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-5220
TitleThe architecture of the DNA polymerase-PCNA-DNA ternary complex
Map dataThis is a map of PolB-PCNA-DNA complex
Sample
  • Sample: Pyrococcus furiosus PolB-PCNA-DNA
  • Protein or peptide: DNA polymerase
  • Protein or peptide: proliferating cell nuclear antigen
  • DNA: DNA
KeywordsDNA replication / DNA clamp
Biological speciesPyrococcus furiosus (archaea) / unidentified (others)
Methodsingle particle reconstruction / Resolution: 19.0 Å
AuthorsMayanagi K / Nishida H / Kiyonari S / Saito M / Kohda D / Ishino Y / Shirai T / Morikawa K
CitationJournal: Proc Natl Acad Sci U S A / Year: 2011
Title: Architecture of the DNA polymerase B-proliferating cell nuclear antigen (PCNA)-DNA ternary complex.
Authors: Kouta Mayanagi / Shinichi Kiyonari / Hirokazu Nishida / Mihoko Saito / Daisuke Kohda / Yoshizumi Ishino / Tsuyoshi Shirai / Kosuke Morikawa /
Abstract: DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This ...DNA replication in archaea and eukaryotes is executed by family B DNA polymerases, which exhibit full activity when complexed with the DNA clamp, proliferating cell nuclear antigen (PCNA). This replication enzyme consists of the polymerase and exonuclease moieties responsible for DNA synthesis and editing (proofreading), respectively. Because of the editing activity, this enzyme ensures the high fidelity of DNA replication. However, it remains unclear how the PCNA-complexed enzyme temporally switches between the polymerizing and editing modes. Here, we present the three-dimensional structure of the Pyrococcus furiosus DNA polymerase B-PCNA-DNA ternary complex, which is the core component of the replisome, determined by single particle electron microscopy of negatively stained samples. This structural view, representing the complex in the editing mode, revealed the whole domain configuration of the trimeric PCNA ring and the DNA polymerase, including protein-protein and protein-DNA contacts. Notably, besides the authentic DNA polymerase-PCNA interaction through a PCNA-interacting protein (PIP) box, a novel contact was found between DNA polymerase and the PCNA subunit adjacent to that with the PIP contact. This contact appears to be responsible for the configuration of the complex specific for the editing mode. The DNA was located almost at the center of PCNA and exhibited a substantial and particular tilt angle against the PCNA ring plane. The obtained molecular architecture of the complex, including the new contact found in this work, provides clearer insights into the switching mechanism between the two distinct modes, thus highlighting the functional significance of PCNA in the replication process.
History
DepositionJul 23, 2010-
Header (metadata) releaseDec 8, 2010-
Map releaseAug 22, 2012-
UpdateAug 22, 2012-
Current statusAug 22, 2012Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2.25
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 2.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5220_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5220.map.gz / Format: CCP4 / Size: 670.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a map of PolB-PCNA-DNA complex
Voxel sizeX=Y=Z: 3.1 Å
Density
Contour LevelBy AUTHOR: 2.25 / Movie #1: 2.25
Minimum - Maximum-3.8578527 - 8.5686512
Average (Standard dev.)-0.00000002 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-9-9-9
Dimensions565656
Spacing565656
CellA=B=C: 173.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.13.13.1
M x/y/z565656
origin x/y/z0.0000.0000.000
length x/y/z173.600173.600173.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-99-99-99
NX/NY/NZ200200200
MAP C/R/S123
start NC/NR/NS-9-9-9
NC/NR/NS565656
D min/max/mean-3.8588.569-0.000

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Supplemental data

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Sample components

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Entire : Pyrococcus furiosus PolB-PCNA-DNA

EntireName: Pyrococcus furiosus PolB-PCNA-DNA
Components
  • Sample: Pyrococcus furiosus PolB-PCNA-DNA
  • Protein or peptide: DNA polymerase
  • Protein or peptide: proliferating cell nuclear antigen
  • DNA: DNA

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Supramolecule #1000: Pyrococcus furiosus PolB-PCNA-DNA

SupramoleculeName: Pyrococcus furiosus PolB-PCNA-DNA / type: sample / ID: 1000 / Details: The sample was monodisperse
Oligomeric state: One PolB binds to one trimer of PCNA and primed DNA
Number unique components: 3
Molecular weightTheoretical: 210 KDa

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Macromolecule #1: DNA polymerase

MacromoleculeName: DNA polymerase / type: protein_or_peptide / ID: 1 / Name.synonym: PolB / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightTheoretical: 104 KDa

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Macromolecule #2: proliferating cell nuclear antigen

MacromoleculeName: proliferating cell nuclear antigen / type: protein_or_peptide / ID: 2 / Name.synonym: PCNA / Oligomeric state: trimer / Recombinant expression: Yes / Database: NCBI
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightTheoretical: 104 KDa

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Macromolecule #3: DNA

MacromoleculeName: DNA / type: dna / ID: 3 / Name.synonym: DNA / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: unidentified (others)
Molecular weightTheoretical: 23 KDa

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Experimental details

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Structure determination

Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.02 mg/mL
BufferpH: 8 / Details: 50 mM Tris-HCl,5 mM MgCl2
GridDetails: 200 mesh Cu grid with carbon support film
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1010
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 400000
Sample stageSpecimen holder: Side entry standard / Specimen holder model: JEOL
Alignment procedureLegacy - Astigmatism: objective astigmatism was corrected using a quadrupole stigmator at 400,000 times magnification.
Detailsminimum dose
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN

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Image processing

Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 19.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN, IMAGIC / Number images used: 18897

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