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Yorodumi- EMDB-2029: Repair complexes of FEN1, DNA and Rad9-Hus1-Rad1 are distinguishe... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2029 | |||||||||
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Title | Repair complexes of FEN1, DNA and Rad9-Hus1-Rad1 are distinguished from their PCNA counterparts by functionally important stability | |||||||||
Map data | image of a surface rendered top-view of human FEN1-911 complex | |||||||||
Sample |
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Keywords | flap endonuclease 1 / processivity clamp / DNA replication and repair / checkpoint clamp / electron microscopy | |||||||||
Biological species | Homo sapiens (human) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 18.0 Å | |||||||||
Authors | Querol-Audi J / Yan C / Xu X / Tsutakawa SE / Tsai M / Tainer JA / Cooper PK / Nogales E / Ivanov I | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_2029.map.gz | 2 MB | EMDB map data format | |
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Header (meta data) | emd-2029-v30.xml emd-2029.xml | 11.8 KB 11.8 KB | Display Display | EMDB header |
Images | emd-2029.png | 41.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2029 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2029 | HTTPS FTP |
-Validation report
Summary document | emd_2029_validation.pdf.gz | 193.2 KB | Display | EMDB validaton report |
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Full document | emd_2029_full_validation.pdf.gz | 192.3 KB | Display | |
Data in XML | emd_2029_validation.xml.gz | 5.4 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2029 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2029 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2029.map.gz / Format: CCP4 / Size: 3.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | image of a surface rendered top-view of human FEN1-911 complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.8 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Human FEN1/911/DNA ternary complex
Entire | Name: Human FEN1/911/DNA ternary complex |
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Components |
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-Supramolecule #1000: Human FEN1/911/DNA ternary complex
Supramolecule | Name: 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 |
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Molecular weight | Experimental: 140 KDa / Theoretical: 140 KDa |
-Macromolecule #1: human checkpoint clamp
Macromolecule | Name: human checkpoint clamp / type: protein_or_peptide / ID: 1 / Name.synonym: h9-1-1 / Number of copies: 1 / Oligomeric state: heterotrimer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Experimental: 100 KDa / Theoretical: 100 KDa |
Recombinant expression | Organism: Insect cells |
-Macromolecule #3: flap endonuclease 1
Macromolecule | Name: flap endonuclease 1 / type: protein_or_peptide / ID: 3 / Name.synonym: FEN1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes |
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Source (natural) | Organism: Homo sapiens (human) / synonym: Human |
Molecular weight | Experimental: 42 KDa / Theoretical: 42 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Macromolecule #2: DNA
Macromolecule | Name: DNA / type: dna / ID: 2 / Name.synonym: double flap DNA / Classification: DNA / Structure: DOUBLE HELIX / Synthetic?: Yes |
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Source (natural) | Organism: synthetic construct (others) |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL |
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Buffer | pH: 7.6 / Details: 20mM Hepes, 80mM KCl, 1mM MgCl2 |
Staining | Type: NEGATIVE Details: sample adsorbed on continuous carbon and stained with 2% w/v uranyl acetate |
Grid | Details: 200 mesh copper grid |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Temperature | Average: 297 K |
Alignment procedure | Legacy - Astigmatism: objective lens astigmatism was corrected at 100,000 times magnification |
Image recording | Category: CCD / Film or detector model: GENERIC GATAN (4k x 4k) / Average electron dose: 20 e/Å2 |
Electron beam | Acceleration voltage: 120 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 80000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal defocus max: 1.7 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 80000 |
Sample stage | Specimen holder: eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC |
-Image processing
Details | The particles were selected using an automatic selection program |
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CTF correction | Details: whole image |
Final reconstruction | Applied 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 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: MDFF method |
Details | Protocol: flexible fitting. complex was refined by flexible fitting into the EM map using the MDFF method. DNA was not included. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
-Atomic model buiding 2
Initial model | PDB ID: |
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Software | Name: MDFF method |
Details | Protocol: flexible fitting. complex was refined by flexible fitting into the EM map using the MDFF method. DNA was not included. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |