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Yorodumi- PDB-5a9k: Structural basis for DNA strand separation by a hexameric replica... -
+Open data
-Basic information
Entry | Database: PDB / ID: 5a9k | ||||||
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Title | Structural basis for DNA strand separation by a hexameric replicative helicase | ||||||
Components | REPLICATION PROTEIN E1 | ||||||
Keywords | HYDROLASE / DNA REPLICATION FORK | ||||||
Function / homology | Function and homology information DNA helicase activity / DNA replication / DNA helicase / host cell nucleus / ATP hydrolysis activity / DNA binding / ATP binding Similarity search - Function | ||||||
Biological species | BOVINE PAPILLOMAVIRUS | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 19 Å | ||||||
Authors | Chaban, Y. / Stead, J.A. / Ryzhenkova, K. / Whelan, F. / Lamber, K. / Antson, F. / Sanders, C.M. / Orlova, E.V. | ||||||
Citation | Journal: Nucleic Acids Res / Year: 2015 Title: Structural basis for DNA strand separation by a hexameric replicative helicase. Authors: Yuriy Chaban / Jonathan A Stead / Ksenia Ryzhenkova / Fiona Whelan / Ekaterina P Lamber / Alfred Antson / Cyril M Sanders / Elena V Orlova / Abstract: Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined ...Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted 'steric exclusion' model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5a9k.cif.gz | 321.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5a9k.ent.gz | 260 KB | Display | PDB format |
PDBx/mmJSON format | 5a9k.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5a9k_validation.pdf.gz | 749 KB | Display | wwPDB validaton report |
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Full document | 5a9k_full_validation.pdf.gz | 771.5 KB | Display | |
Data in XML | 5a9k_validation.xml.gz | 47.3 KB | Display | |
Data in CIF | 5a9k_validation.cif.gz | 70.9 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/a9/5a9k ftp://data.pdbj.org/pub/pdb/validation_reports/a9/5a9k | HTTPS FTP |
-Related structure data
Related structure data | 3087MC 3088C 3089C 3090C 3091C 5a9l 5a9m 5a9n 5a9o M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 34645.016 Da / Num. of mol.: 6 / Fragment: HELICASE DOMAIN, RESIDUES 301-605 Source method: isolated from a genetically manipulated source Source: (gene. exp.) BOVINE PAPILLOMAVIRUS / Organ: NUCLEUS / Plasmid: PET11C / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P03116, DNA helicase #2: Chemical | ChemComp-PO4 / #3: Chemical | #4: Water | ChemComp-HOH / | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: HELICASE DOMAIN OF THE FULL-LENGTH E1 HELICASE FROM BOVINE PAPILLOMAVIRUS Type: VIRUS |
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Buffer solution | Name: 10 MM TRIS-CL PH 8.0, 225 MM NACL, 2 MM DTT, 0.1 MM PMSF, 0.1 MM EDTA pH: 8 Details: 10 MM TRIS-CL PH 8.0, 225 MM NACL, 2 MM DTT, 0.1 MM PMSF, 0.1 MM EDTA |
Specimen | Conc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO |
EM staining | Type: NEGATIVE / Material: uranyl acetate |
Specimen support | Details: CARBON |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
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Microscopy | Model: FEI TECNAI F20 / Date: Feb 18, 2010 |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 62000 X / Calibrated magnification: 67000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.1 mm |
Specimen holder | Temperature: 293 K |
Image recording | Electron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) |
Image scans | Num. digital images: 30 |
Radiation wavelength | Relative weight: 1 |
-Processing
EM software |
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CTF correction | Details: FRAMES | ||||||||||||||||||||
Symmetry | Point symmetry: C6 (6 fold cyclic) | ||||||||||||||||||||
3D reconstruction | Method: FILTERED BACK PROJECTION / Resolution: 19 Å / Num. of particles: 8265 / Actual pixel size: 1.6 Å Magnification calibration: CROSS- -CORRELATION DENSITIES WITHIN SPHERICAL SHELL Details: SUBMISSION BASED ON EXPERIMENTAL DATA FROM EMDB EMD-3087. (DEPOSITION ID: 13536). Symmetry type: POINT | ||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient Details: METHOD--LOCAL CORRELATION REFINEMENT PROTOCOL--X-RAY | ||||||||||||||||||||
Atomic model building | PDB-ID: 2V9P Accession code: 2V9P / Source name: PDB / Type: experimental model | ||||||||||||||||||||
Refinement | Highest resolution: 19 Å | ||||||||||||||||||||
Refinement step | Cycle: LAST / Highest resolution: 19 Å
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