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- PDB-5a9k: Structural basis for DNA strand separation by a hexameric replica... -

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Basic information

Entry
Database: PDB / ID: 5a9k
TitleStructural basis for DNA strand separation by a hexameric replicative helicase
ComponentsREPLICATION PROTEIN E1DNA replication
KeywordsHYDROLASE / DNA REPLICATION FORK
Function / homology
Function and homology information


DNA helicase activity / DNA helicase / DNA replication / host cell nucleus / ATP hydrolysis activity / DNA binding / ATP binding
Similarity search - Function
DNA helicase E1, C-terminal, Papillomavirus / DNA helicase E1, N-terminal, Papillomavirus / Replication protein E1, papillomavirus / DNA helicase E1, DNA-binding domain, papillomavirus / DNA helicase E1, DNA-binding domain superfamily, papillomavirus / Papillomavirus helicase / E1 Protein, N terminal domain / Papillomavirus E1, DNA-binding domain / Zinc finger, large T-antigen D1 domain superfamily / Helicase, superfamily 3, DNA virus ...DNA helicase E1, C-terminal, Papillomavirus / DNA helicase E1, N-terminal, Papillomavirus / Replication protein E1, papillomavirus / DNA helicase E1, DNA-binding domain, papillomavirus / DNA helicase E1, DNA-binding domain superfamily, papillomavirus / Papillomavirus helicase / E1 Protein, N terminal domain / Papillomavirus E1, DNA-binding domain / Zinc finger, large T-antigen D1 domain superfamily / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHATE ION / Replication protein E1
Similarity search - Component
Biological speciesBOVINE PAPILLOMAVIRUS
MethodELECTRON MICROSCOPY / single particle reconstruction / negative staining / Resolution: 19 Å
AuthorsChaban, Y. / Stead, J.A. / Ryzhenkova, K. / Whelan, F. / Lamber, K. / Antson, F. / Sanders, C.M. / Orlova, E.V.
CitationJournal: 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
DepositionJul 21, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 26, 2015Provider: repository / Type: Initial release
Revision 1.1Oct 7, 2015Group: Database references
Revision 1.2Aug 2, 2017Group: Data collection / Refinement description / Category: em_3d_fitting / em_software
Item: _em_3d_fitting.target_criteria / _em_software.fitting_id ..._em_3d_fitting.target_criteria / _em_software.fitting_id / _em_software.image_processing_id / _em_software.name

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Structure visualization

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  • Deposited structure unit
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  • EMDB-3087
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: REPLICATION PROTEIN E1
B: REPLICATION PROTEIN E1
C: REPLICATION PROTEIN E1
D: REPLICATION PROTEIN E1
E: REPLICATION PROTEIN E1
F: REPLICATION PROTEIN E1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)208,79818
Polymers207,8706
Non-polymers92812
Water32418
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
MethodPISA

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Components

#1: Protein
REPLICATION PROTEIN E1 / DNA replication / 3.6.4.12 / ATP-DEPENDENT HELICASE E1


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: NUCLEUSCell nucleus / Plasmid: PET11C / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P03116, DNA helicase
#2: Chemical
ChemComp-PO4 / PHOSPHATE ION / Phosphate


Mass: 94.971 Da / Num. of mol.: 9 / Source method: obtained synthetically / Formula: PO4
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: HELICASE DOMAIN OF THE FULL-LENGTH E1 HELICASE FROM BOVINE PAPILLOMAVIRUS
Type: VIRUS
Buffer solutionName: 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
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: YES / Vitrification applied: NO
EM stainingType: NEGATIVE / Material: uranyl acetate
Specimen supportDetails: CARBON

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Electron microscopy imaging

Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company
MicroscopyModel: FEI TECNAI F20 / Date: Feb 18, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 62000 X / Calibrated magnification: 67000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.1 mm
Specimen holderTemperature: 293 K
Image recordingElectron dose: 20 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 30
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1CTFFIND3CTF correction
2ctfitCTF correction
3UCSF Chimeramodel fitting
4IMAGIC3D reconstruction
CTF correctionDetails: FRAMES
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionMethod: 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 buildingProtocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Details: METHOD--LOCAL CORRELATION REFINEMENT PROTOCOL--X-RAY
Atomic model buildingPDB-ID: 2V9P

2v9p

RefinementHighest resolution: 19 Å
Refinement stepCycle: LAST / Highest resolution: 19 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms12993 0 48 18 13059

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