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- EMDB-2029: Repair complexes of FEN1, DNA and Rad9-Hus1-Rad1 are distinguishe... -

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Basic information

Entry
Database: EMDB / ID: EMD-2029
TitleRepair complexes of FEN1, DNA and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability
Map dataimage of a surface rendered top-view of human FEN1-911 complex
Sample
  • Sample: Human FEN1/911/DNA ternary complex
  • Protein or peptide: human checkpoint clamp
  • Protein or peptide: flap endonuclease 1Flap structure-specific endonuclease 1
  • DNA: DNA
Keywordsflap endonuclease 1 / processivity clamp / DNA replication and repair / checkpoint clamp / electron microscopy
Biological speciesHomo sapiens (human) / synthetic construct (others)
Methodsingle particle reconstruction / negative staining / Resolution: 18.0 Å
AuthorsQuerol-Audi J / Yan C / Xu X / Tsutakawa SE / Tsai M / Tainer JA / Cooper PK / Nogales E / Ivanov I
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Repair complexes of FEN1 endonuclease, DNA, and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability.
Authors: Jordi Querol-Audí / Chunli Yan / Xiaojun Xu / Susan E Tsutakawa / Miaw-Sheue Tsai / John A Tainer / Priscilla K Cooper / Eva Nogales / Ivaylo Ivanov /
Abstract: Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins ...Processivity clamps such as proliferating cell nuclear antigen (PCNA) and the checkpoint sliding clamp Rad9/Rad1/Hus1 (9-1-1) act as versatile scaffolds in the coordinated recruitment of proteins involved in DNA replication, cell-cycle control, and DNA repair. Association and handoff of DNA-editing enzymes, such as flap endonuclease 1 (FEN1), with sliding clamps are key processes in biology, which are incompletely understood from a mechanistic point of view. We have used an integrative computational and experimental approach to define the assemblies of FEN1 with double-flap DNA substrates and either proliferating cell nuclear antigen or the checkpoint sliding clamp 9-1-1. Fully atomistic models of these two ternary complexes were developed and refined through extensive molecular dynamics simulations to expose their conformational dynamics. Clustering analysis revealed the most dominant conformations accessible to the complexes. The cluster centroids were subsequently used in conjunction with single-particle electron microscopy data to obtain a 3D EM reconstruction of the human 9-1-1/FEN1/DNA assembly at 18-Å resolution. Comparing the structures of the complexes revealed key differences in the orientation and interactions of FEN1 and double-flap DNA with the two clamps that are consistent with their respective functions in providing inherent flexibility for lagging strand DNA replication or inherent stability for DNA repair.
History
DepositionJan 11, 2012-
Header (metadata) releaseJan 27, 2012-
Map releaseSep 26, 2012-
UpdateOct 17, 2012-
Current statusOct 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 4.6
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd-2029.png

Downloads & links

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Map

FileDownload / File: emd_2029.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationimage of a surface rendered top-view of human FEN1-911 complex
Voxel sizeX=Y=Z: 2.8 Å
Density
Contour LevelBy AUTHOR: 4.6 / Movie #1: 4.6
Minimum - Maximum-6.37851381 - 18.357995989999999
Average (Standard dev.)0.0 (±0.99999952)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions969696
Spacing969696
CellA=B=C: 268.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.82.82.8
M x/y/z969696
origin x/y/z0.0000.0000.000
length x/y/z268.800268.800268.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS969696
D min/max/mean-6.37918.3580.000

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Supplemental data

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Sample components

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Entire : Human FEN1/911/DNA ternary complex

EntireName: Human FEN1/911/DNA ternary complex
Components
  • Sample: Human FEN1/911/DNA ternary complex
  • Protein or peptide: human checkpoint clamp
  • Protein or peptide: flap endonuclease 1Flap structure-specific endonuclease 1
  • DNA: DNA

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Supramolecule #1000: Human FEN1/911/DNA ternary complex

SupramoleculeName: Human FEN1/911/DNA ternary complex / type: sample / ID: 1000
Oligomeric state: One FEN1 binds to one 911 and one DNA substrate
Number unique components: 3
Molecular weightExperimental: 140 KDa / Theoretical: 140 KDa

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Macromolecule #1: human checkpoint clamp

MacromoleculeName: human checkpoint clamp / type: protein_or_peptide / ID: 1 / Name.synonym: h9-1-1 / Number of copies: 1 / Oligomeric state: heterotrimer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 100 KDa / Theoretical: 100 KDa
Recombinant expressionOrganism: Insect cells

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Macromolecule #3: flap endonuclease 1

MacromoleculeName: flap endonuclease 1 / type: protein_or_peptide / ID: 3 / Name.synonym: FEN1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Molecular weightExperimental: 42 KDa / Theoretical: 42 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: DNA

MacromoleculeName: DNA / type: dna / ID: 2 / Name.synonym: double flap 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

Concentration1 mg/mL
BufferpH: 7.6 / Details: 20mM Hepes, 80mM KCl, 1mM MgCl2
StainingType: NEGATIVE
Details: sample adsorbed on continuous carbon and stained with 2% w/v uranyl acetate
GridDetails: 200 mesh copper grid
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 80000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000
Sample stageSpecimen holder: eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC
TemperatureAverage: 297 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification
Image recordingCategory: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Average electron dose: 20 e/Å2

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Image processing

CTF correctionDetails: whole image
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN2 / Number images used: 12000
DetailsThe particles were selected using an automatic selection program

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Atomic model buiding 1

Initial modelPDB ID:

3q8l

SoftwareName: MDFF method
DetailsProtocol: flexible fitting. complex was refined by flexible fitting into the EM map using the MDFF method. DNA was not included.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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Atomic model buiding 2

Initial modelPDB ID:

1ul1

SoftwareName: MDFF method
DetailsProtocol: flexible fitting. complex was refined by flexible fitting into the EM map using the MDFF method. DNA was not included.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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