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- PDB-5fku: cryo-EM structure of the E. coli replicative DNA polymerase compl... -

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Basic information

Entry
Database: PDB / ID: 5fku
Titlecryo-EM structure of the E. coli replicative DNA polymerase complex in DNA free state (DNA polymerase III alpha, beta, epsilon, tau complex)
Components
  • DNA POLYMERASE III SUBUNIT ALPHADNA polymerase III holoenzyme
  • DNA POLYMERASE III SUBUNIT BETADNA polymerase III holoenzyme
  • DNA POLYMERASE III SUBUNIT EPSILONDNA polymerase III holoenzyme
  • DNA POLYMERASE III SUBUNIT TAUDNA polymerase III holoenzyme
KeywordsTRANSFERASE / DNA REPLICATION / DNA POLYMERASE III ALPHA / DNA POLYMERASE III BETA / DNA POLYMERASE III EPSILON / DNA POLYMERASE III TAU
Function / homology
Function and homology information


DNA polymerase III, core complex / DNA polymerase III, clamp loader complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA replication proofreading / DNA polymerase III complex / lagging strand elongation / replisome / regulation of DNA-templated DNA replication initiation ...DNA polymerase III, core complex / DNA polymerase III, clamp loader complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA replication proofreading / DNA polymerase III complex / lagging strand elongation / replisome / regulation of DNA-templated DNA replication initiation / exonuclease activity / DNA strand elongation involved in DNA replication / leading strand elongation / DNA polymerase processivity factor activity / error-prone translesion synthesis / negative regulation of DNA-templated DNA replication initiation / 3'-5' exonuclease activity / ribonucleoside triphosphate phosphatase activity / DNA-templated DNA replication / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA damage response / ATP hydrolysis activity / protein homodimerization activity / DNA binding / ATP binding / metal ion binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
: / DNA polymerase III subunit alpha, C-terminal domain / DNA polymerase 3, epsilon subunit / DNA polymerase III epsilon subunit, exonuclease domain / Bacterial DNA polymerase III alpha subunit, thumb domain / DNA polymerase III, alpha subunit / Bacterial DNA polymerase III, alpha subunit, NTPase domain / DNA polymerase, helix-hairpin-helix motif / DNA polymerase III alpha subunit finger domain / Bacterial DNA polymerase III alpha NTPase domain ...: / DNA polymerase III subunit alpha, C-terminal domain / DNA polymerase 3, epsilon subunit / DNA polymerase III epsilon subunit, exonuclease domain / Bacterial DNA polymerase III alpha subunit, thumb domain / DNA polymerase III, alpha subunit / Bacterial DNA polymerase III, alpha subunit, NTPase domain / DNA polymerase, helix-hairpin-helix motif / DNA polymerase III alpha subunit finger domain / Bacterial DNA polymerase III alpha NTPase domain / Helix-hairpin-helix motif / Bacterial DNA polymerase III alpha subunit finger domain / DNA polymerase III, tau subunit, domain V / DNA polymerase III subunit tau, DnaB-binding domain IV / DNA polymerase III, tau subunit, domain V superfamily / DNA polymerase III, subunit gamma/tau, helical lid domain / DNA polymerase III subunits tau domain IV DnaB-binding / DNA polymerase III tau subunit V interacting with alpha / DNA polymerase III, subunit gamma/ tau, N-terminal / DNA polymerase III, gamma subunit, domain III / DNA polymerase III subunits gamma and tau domain III / PHP domain / PHP domain / Polymerase/histidinol phosphatase, N-terminal / DNA polymerase alpha chain like domain / Polymerase/histidinol phosphatase-like / DNA polymerase III, delta subunit / Exonuclease / DNA polymerase III, beta sliding clamp / DNA polymerase III, beta sliding clamp, N-terminal / DNA polymerase III, beta sliding clamp, C-terminal / DNA polymerase III, beta sliding clamp, central / DNA polymerase III beta subunit, N-terminal domain / DNA polymerase III beta subunit, central domain / DNA polymerase III beta subunit, C-terminal domain / DNA polymerase III beta subunit / DNA polymerase III, clamp loader complex, gamma/delta/delta subunit, C-terminal / Exonuclease, RNase T/DNA polymerase III / EXOIII / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / ClpA/B family / : / Ribonuclease H superfamily / Ribonuclease H-like superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA polymerase III subunit epsilon / DNA polymerase III subunit tau / Beta sliding clamp / DNA polymerase III subunit alpha
Similarity search - Component
Biological speciesESCHERICHIA COLI K-12 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 8.34 Å
AuthorsFernandez-Leiro, R. / Conrad, J. / Scheres, S.H.W. / Lamers, M.H.
CitationJournal: Elife / Year: 2015
Title: cryo-EM structures of the replicative DNA polymerase reveal its dynamic interactions with the DNA sliding clamp, exonuclease and .
Authors: Rafael Fernandez-Leiro / Julian Conrad / Sjors Hw Scheres / Meindert H Lamers /
Abstract: The replicative DNA polymerase PolIIIα from is a uniquely fast and processive enzyme. For its activity it relies on the DNA sliding clamp β, the proofreading exonuclease ε and the C-terminal ...The replicative DNA polymerase PolIIIα from is a uniquely fast and processive enzyme. For its activity it relies on the DNA sliding clamp β, the proofreading exonuclease ε and the C-terminal domain of the clamp loader subunit τ. Due to the dynamic nature of the four-protein complex it has long been refractory to structural characterization. Here we present the 8 Å resolution cryo-electron microscopy structures of DNA-bound and DNA-free states of the PolIII-clamp-exonuclease-τ complex. The structures show how the polymerase is tethered to the DNA through multiple contacts with the clamp and exonuclease. A novel contact between the polymerase and clamp is made in the DNA bound state, facilitated by a large movement of the polymerase tail domain and τ. These structures provide crucial insights into the organization of the catalytic core of the replisome and form an important step towards determining the structure of the complete holoenzyme.
History
DepositionOct 20, 2015Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 25, 2015Provider: repository / Type: Initial release
Revision 1.1Dec 21, 2016Group: Database references
Revision 1.2Apr 19, 2017Group: Other
Revision 1.3Feb 27, 2019Group: Data collection / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / pdbx_database_proc / struct_conn
Item: _citation.title / _citation_author.identifier_ORCID / _struct_conn.pdbx_leaving_atom_flag
Revision 1.4May 8, 2019Group: Advisory / Data collection / Derived calculations
Category: pdbx_data_processing_status / pdbx_validate_close_contact ...pdbx_data_processing_status / pdbx_validate_close_contact / struct_conn / struct_conn_type

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Structure visualization

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Assembly

Deposited unit
A: DNA POLYMERASE III SUBUNIT ALPHA
B: DNA POLYMERASE III SUBUNIT BETA
C: DNA POLYMERASE III SUBUNIT BETA
D: DNA POLYMERASE III SUBUNIT EPSILON
E: DNA POLYMERASE III SUBUNIT TAU


Theoretical massNumber of molelcules
Total (without water)254,7535
Polymers254,7535
Non-polymers00
Water0
1


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

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Components

#1: Protein DNA POLYMERASE III SUBUNIT ALPHA / DNA polymerase III holoenzyme


Mass: 130088.430 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI K-12 (bacteria) / Plasmid: PET3D / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P10443, DNA-directed DNA polymerase
#2: Protein DNA POLYMERASE III SUBUNIT BETA / DNA polymerase III holoenzyme / BETA SLIDING CLAMP / BETA CLAMP


Mass: 40630.508 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI K-12 (bacteria) / Plasmid: PET3D / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P0A988, DNA-directed DNA polymerase
#3: Protein DNA POLYMERASE III SUBUNIT EPSILON / DNA polymerase III holoenzyme


Mass: 27118.984 Da / Num. of mol.: 1 / Mutation: YES
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI K-12 (bacteria) / Plasmid: PET3D / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P03007, DNA-directed DNA polymerase
#4: Protein DNA POLYMERASE III SUBUNIT TAU / DNA polymerase III holoenzyme / DNA POLYMERASE III SUBUNIT GAMMA


Mass: 16284.270 Da / Num. of mol.: 1
Fragment: POLYMERASE-BINDING DOMAIN OF TAU, UNP RESIDUES 500-643
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) ESCHERICHIA COLI K-12 (bacteria) / Plasmid: PET3D / Production host: ESCHERICHIA COLI (E. coli) / Strain (production host): BL21(DE3) / References: UniProt: P06710, DNA-directed DNA polymerase
Compound detailsENGINEERED RESIDUE IN CHAIN A, ALA 921 TO LEU ENGINEERED RESIDUE IN CHAIN A, MET 923 TO LEU ...ENGINEERED RESIDUE IN CHAIN A, ALA 921 TO LEU ENGINEERED RESIDUE IN CHAIN A, MET 923 TO LEU ENGINEERED RESIDUE IN CHAIN D, THR 183 TO LEU ENGINEERED RESIDUE IN CHAIN D, MET 185 TO LEU ENGINEERED RESIDUE IN CHAIN D, ALA 186 TO PRO ENGINEERED RESIDUE IN CHAIN D, PHE 187 TO LEU

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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: DNA POLYMERASE III CATALYTIC COMPLEX (ALPHA, EPSILON, BETA, TAU)
Type: COMPLEX
Buffer solutionName: 25 MM HEPES PH 7.5, 150 MM NACL, AND 2 MM DTT / pH: 7.5 / Details: 25 MM HEPES PH 7.5, 150 MM NACL, AND 2 MM DTT
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: HOLEY CARBON
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Details: LIQUID ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: May 12, 2014
Details: TITAN KRIOS GOOD MICROGRAPHS WERE SELECTED FOR DIGITISATION
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 X / Calibrated magnification: 28409 X / Nominal defocus max: 4000 nm / Nominal defocus min: 2000 nm / Cs: 2.7 mm
Specimen holderTemperature: 85 K
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K2 QUANTUM (4k x 4k)
Image scansNum. digital images: 1350

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Processing

SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 8.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 16970 / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
RefinementHighest resolution: 8.34 Å
Refinement stepCycle: LAST / Highest resolution: 8.34 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms17387 0 0 0 17387

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