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

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Basic information

Entry
Database: PDB / ID: 5fkw
Titlecryo-EM structure of the E. coli replicative DNA polymerase complex bound to DNA (DNA polymerase III alpha, beta, epsilon)
Components
  • (PRIMER-TEMPLATE DUPLEX DNA) x 2
  • DNA POLYMERASE III ALPHA
  • DNA POLYMERASE III BETA
  • DNA POLYMERASE III EPSILON
KeywordsTRANSFERASE / DNA REPLICATION / DNA POLYMERASE III ALPHA / DNA POLYMERASE III BETA / DNA POLYMERASE III EPSILON
Function / homology
Function and homology information


DNA polymerase III, core complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA polymerase III complex / DNA replication proofreading / lagging strand elongation / replisome / regulation of DNA-templated DNA replication initiation / exonuclease activity ...DNA polymerase III, core complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA polymerase III complex / DNA replication proofreading / lagging strand elongation / replisome / regulation of DNA-templated DNA replication initiation / exonuclease activity / DNA strand elongation involved in DNA replication / leading strand elongation / error-prone translesion synthesis / 3'-5' exonuclease activity / negative regulation of DNA-templated DNA replication initiation / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA damage response / protein homodimerization activity / DNA 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 ...: / : / 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 / PHP domain / PHP domain / Polymerase/histidinol phosphatase, N-terminal / DNA polymerase alpha chain like domain / Polymerase/histidinol phosphatase-like / 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 / Exonuclease / Exonuclease, RNase T/DNA polymerase III / EXOIII / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / OB-fold nucleic acid binding domain / : / Ribonuclease H superfamily / Ribonuclease H-like superfamily / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA polymerase III subunit epsilon / Beta sliding clamp / DNA polymerase III subunit alpha
Similarity search - Component
Biological speciesESCHERICHIA COLI K-12 (bacteria)
SYNTHETIC CONSTRUCT (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 7.3 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
Category: citation / citation_author ...citation / citation_author / pdbx_database_proc / pdbx_seq_map_depositor_info
Item: _citation.title / _citation_author.identifier_ORCID / _pdbx_seq_map_depositor_info.one_letter_code_mod
Revision 1.4May 8, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: DNA POLYMERASE III ALPHA
B: DNA POLYMERASE III BETA
C: DNA POLYMERASE III BETA
D: DNA POLYMERASE III EPSILON
P: PRIMER-TEMPLATE DUPLEX DNA
T: PRIMER-TEMPLATE DUPLEX DNA


Theoretical massNumber of molelcules
Total (without water)255,0626
Polymers255,0626
Non-polymers00
Water00
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 ALPHA


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 BETA


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 EPSILON


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: DNA chain PRIMER-TEMPLATE DUPLEX DNA


Mass: 7797.048 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: TEMPLATE STRAND TCAGGAGTCCTTCGTCCTAGTACTACTCC PRIMER STRAND GGAGTAGTACTAGGACGAAGGACTC
Source: (synth.) SYNTHETIC CONSTRUCT (others)
#5: DNA chain PRIMER-TEMPLATE DUPLEX DNA


Mass: 8796.659 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: TEMPLATE STRAND TCAGGAGTCCTTCGTCCTAGTACTACTCC PRIMER STRAND GGAGTAGTACTAGGACGAAGGACTC
Source: (synth.) SYNTHETIC CONSTRUCT (others)

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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
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / 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: 7.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40582 / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: POINT
RefinementHighest resolution: 7.3 Å
Refinement stepCycle: LAST / Highest resolution: 7.3 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms14679 1047 0 0 15726

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