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Yorodumi- EMDB-2030: Repair complexes of FEN1, DNA and Rad9-Hus1-Rad1 are distinguishe... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-2030 | |||||||||
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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 | surface rendered top-view of human 9-1-1 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) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 17.0 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_2030.map.gz | 1.8 MB | EMDB map data format | |
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Header (meta data) | emd-2030-v30.xml emd-2030.xml | 11.5 KB 11.5 KB | Display Display | EMDB header |
Images | emd-2030.png | 48 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-2030 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-2030 | HTTPS FTP |
-Validation report
Summary document | emd_2030_validation.pdf.gz | 194.6 KB | Display | EMDB validaton report |
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Full document | emd_2030_full_validation.pdf.gz | 193.7 KB | Display | |
Data in XML | emd_2030_validation.xml.gz | 5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2030 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-2030 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_2030.map.gz / Format: CCP4 / Size: 2.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | surface rendered top-view of human 9-1-1 complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.54 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 911 complex
Entire | Name: human 911 complex |
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Components |
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-Supramolecule #1000: human 911 complex
Supramolecule | Name: human 911 complex / type: sample / ID: 1000 / Oligomeric state: heterotrimer / Number unique components: 3 |
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Molecular weight | Experimental: 100 KDa / Theoretical: 100 KDa |
-Macromolecule #1: Rad9
Macromolecule | Name: Rad9 / 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: 43 KDa / Theoretical: 43 KDa |
-Macromolecule #2: Rad1
Macromolecule | Name: Rad1 / type: protein_or_peptide / ID: 2 / 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: 31 KDa / Theoretical: 31 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Macromolecule #3: Hus1
Macromolecule | Name: Hus1 / type: protein_or_peptide / ID: 3 / 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: 32 KDa / Theoretical: 32 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 8 / Details: 20mM Hepes, 150mM NaCl, 3% trehalose |
Staining | Type: NEGATIVE Details: sample adsorbed on continuous carbon and stained with 2% w/v PTA |
Grid | Details: 200 mesh copper grid |
Vitrification | Cryogen name: NONE / Instrument: OTHER |
-Electron microscopy
Microscope | FEI TECNAI 12 |
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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: KODAK SO-163 FILM / Digitization - Sampling interval: 2.54 µm / Number real images: 15 |
Electron beam | Acceleration voltage: 120 kV / Electron source: LAB6 |
Electron optics | Calibrated magnification: 50000 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 6.3 mm / Nominal defocus max: 0.8 µm / Nominal defocus min: 0.4 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: eucentric / Specimen holder model: SIDE ENTRY, EUCENTRIC |
-Image processing
Details | The particles were manually selected using boxer |
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CTF correction | Details: whole image |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 5000 |
-Atomic model buiding 1
Initial model | PDB ID: |
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Software | Name: chimera |
Details | Protocol: rigid body |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |