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- EMDB-1485: The archaeal DNA Ligase-PCNA-DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-1485
TitleThe archaeal DNA Ligase-PCNA-DNA complex
Map dataThis is a map of DNA ligase-PCNA-DNA complex
Sample
  • Sample: Pyrococcus furiosus DNA Ligase bound to PCNA and DNA
  • Protein or peptide: Pyrococcus furiosus DNA Ligase
  • Protein or peptide: Proliferating cell nuclear antigen
  • DNA: PCNA-nicked DNA
Biological speciesPyrococcus furiosus (archaea) / synthetic construct (others)
Methodsingle particle reconstruction / negative staining / Resolution: 17.0 Å
AuthorsMayanagi K / Kiyonari S / Ishino Y / Shirai T / Morikawa K
CitationJournal: Proc Natl Acad Sci U S A / Year: 2009
Title: Mechanism of replication machinery assembly as revealed by the DNA ligase-PCNA-DNA complex architecture.
Authors: Kouta Mayanagi / Shinichi Kiyonari / Mihoko Saito / Tsuyoshi Shirai / Yoshizumi Ishino / Kosuke Morikawa /
Abstract: The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the ...The 3D structure of the ternary complex, consisting of DNA ligase, the proliferating cell nuclear antigen (PCNA) clamp, and DNA, was investigated by single-particle analysis. This report presents the structural view, where the crescent-shaped DNA ligase with 3 distinct domains surrounds the central DNA duplex, encircled by the closed PCNA ring, thus forming a double-layer structure with dual contacts between the 2 proteins. The relative orientations of the DNA ligase domains, which remarkably differ from those of the known crystal structures, suggest that a large domain rearrangement occurs upon ternary complex formation. A second contact was found between the PCNA ring and the middle adenylation domain of the DNA ligase. Notably, the map revealed a substantial DNA tilt from the PCNA ring axis. This structure allows us to propose a switching mechanism for the replication factors operating on the PCNA ring.
History
DepositionFeb 28, 2008-
Header (metadata) releaseFeb 28, 2008-
Map releaseJun 23, 2010-
UpdateOct 31, 2012-
Current statusOct 31, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.19
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.19
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1485.png

Downloads & links

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Map

FileDownload / File: emd_1485.map.gz / Format: CCP4 / Size: 670.9 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a map of DNA ligase-PCNA-DNA complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.1 Å/pix.
x 56 pix.
= 170.5 Å		3.1 Å/pix.
x 56 pix.
= 170.5 Å		3.1 Å/pix.
x 56 pix.
= 170.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 3.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 1.19 / Movie #1: 1.19
Minimum - Maximum-1.62418 - 8.66733
Average (Standard dev.)0.000000000694363 (±0.819917)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-28-28-28
Dimensions565656
Spacing565656
CellA=B=C: 170.5 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.13.13.1
M x/y/z555555
origin x/y/z0.0000.0000.000
length x/y/z170.500170.500170.500
α/β/γ90.00090.00090.000
start NX/NY/NZ-55-55-55
NX/NY/NZ111111111
MAP C/R/S123
start NC/NR/NS-28-28-28
NC/NR/NS565656
D min/max/mean-1.6248.6670.000

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Supplemental data

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Sample components

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Entire : Pyrococcus furiosus DNA Ligase bound to PCNA and DNA

EntireName: Pyrococcus furiosus DNA Ligase bound to PCNA and DNA
Components
  • Sample: Pyrococcus furiosus DNA Ligase bound to PCNA and DNA
  • Protein or peptide: Pyrococcus furiosus DNA Ligase
  • Protein or peptide: Proliferating cell nuclear antigen
  • DNA: PCNA-nicked DNA

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Supramolecule #1000: Pyrococcus furiosus DNA Ligase bound to PCNA and DNA

SupramoleculeName: Pyrococcus furiosus DNA Ligase bound to PCNA and DNA / type: sample / ID: 1000 / Details: The sample was monodisperse / Oligomeric state: Heteropentamer / Number unique components: 3
Molecular weightTheoretical: 160 KDa
Method: One DNA Ligase binds to three PCNA and one nicked DNA

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Macromolecule #1: Pyrococcus furiosus DNA Ligase

MacromoleculeName: Pyrococcus furiosus DNA Ligase / type: protein_or_peptide / ID: 1 / Name.synonym: DNA Ligase / Recombinant expression: No
Source (natural)Organism: Pyrococcus furiosus (archaea)

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Macromolecule #2: Proliferating cell nuclear antigen

MacromoleculeName: Proliferating cell nuclear antigen / type: protein_or_peptide / ID: 2 / Name.synonym: PCNA / Number of copies: 3 / Oligomeric state: Trimeric / Recombinant expression: Yes
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightTheoretical: 28 KDa

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Macromolecule #3: PCNA-nicked DNA

MacromoleculeName: PCNA-nicked DNA / type: dna / ID: 3 / Name.synonym: DNA / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: synthetic construct (others)

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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

Concentration0.02 mg/mL
BufferpH: 6.5 / Details: 20 mM MES,50 mM NaCl,10 mM MgCl2,0.1 mM ATP
StainingType: NEGATIVE
Details: The sample solution was applied to a copper grid supporting a continuous thin-carbon film, left for 1 min, then stained with 3 drops of 2% uranyl acetate, and air dried.
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeJEOL 1010
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 400,000 times magnification
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN / Number real images: 400
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 100 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 5.6 mm / Nominal magnification: 400000
Sample stageSpecimen holder: Eucentric / Specimen holder model: OTHER

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN,IMAGIC / Number images used: 19544

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Atomic model buiding 1

Initial modelPDB ID:

1ge8


Chain - Chain ID: A
DetailsPDBEntryID_givenInChain.
RefinementSpace: REAL

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Atomic model buiding 2

Initial modelPDB ID:

2pol


Chain - Chain ID: A
DetailsPDBEntryID_givenInChain.
RefinementSpace: REAL

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