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- PDB-5m1s: Cryo-EM structure of the E. coli replicative DNA polymerase-clamp... -

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Basic information

Entry
Database: PDB / ID: 5m1s
TitleCryo-EM structure of the E. coli replicative DNA polymerase-clamp-exonuclase-theta complex bound to DNA in the editing mode
Components
  • (DNA polymerase III subunit ...DNA polymerase III holoenzyme) x 4
  • DNA Primer Strand
  • DNA Template Strand
KeywordsDNA BINDING PROTEIN / DNA editing Proofreading Exonuclease Polymerase
Function / homology
Function and homology information


DNA polymerase III, core 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 strand elongation involved in DNA replication ...DNA polymerase III, core 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 strand elongation involved in DNA replication / exonuclease activity / leading strand elongation / error-prone translesion synthesis / negative regulation of DNA-templated DNA replication initiation / 3'-5' exonuclease activity / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA damage response / protein homodimerization activity / DNA binding / identical protein binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
DNA polymerase III-theta, bacterial / DNA polymerase III-theta superfamily / DNA polymerase III, theta subunit / : / : / 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 ...DNA polymerase III-theta, bacterial / DNA polymerase III-theta superfamily / DNA polymerase III, theta subunit / : / : / 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 / 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 / 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 theta / DNA polymerase III subunit alpha
Similarity search - Component
Biological speciesEscherichia coli K12 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsFernandez-Leiro, R. / Conrad, J. / Scheres, S.H.W. / Lamers, M.H.
CitationJournal: Nat Struct Mol Biol / Year: 2017
Title: Self-correcting mismatches during high-fidelity DNA replication.
Authors: Rafael Fernandez-Leiro / Julian Conrad / Ji-Chun Yang / Stefan M V Freund / Sjors H W Scheres / Meindert H Lamers /
Abstract: Faithful DNA replication is essential to all forms of life and depends on the action of 3'-5' exonucleases that remove misincorporated nucleotides from the newly synthesized strand. However, how the ...Faithful DNA replication is essential to all forms of life and depends on the action of 3'-5' exonucleases that remove misincorporated nucleotides from the newly synthesized strand. However, how the DNA is transferred from the polymerase to the exonuclease active site is not known. Here we present the cryo-EM structure of the editing mode of the catalytic core of the Escherichia coli replisome, revealing a dramatic distortion of the DNA whereby the polymerase thumb domain acts as a wedge that separates the two DNA strands. Importantly, NMR analysis of the DNA substrate shows that the presence of a mismatch increases the fraying of the DNA, thus enabling it to reach the exonuclease active site. Therefore the mismatch corrects itself, whereas the exonuclease subunit plays a passive role. Hence, our work provides unique insights into high-fidelity replication and establishes a new paradigm for the correction of misincorporated nucleotides.
History
DepositionOct 10, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 18, 2017Provider: repository / Type: Initial release
Revision 1.1Feb 15, 2017Group: Database references
Revision 1.2Aug 30, 2017Group: Data collection / Derived calculations / Experimental preparation
Category: em_imaging_optics / em_sample_support ...em_imaging_optics / em_sample_support / em_software / struct_conn
Item: _em_imaging_optics.energyfilter_name / _em_sample_support.grid_type ..._em_imaging_optics.energyfilter_name / _em_sample_support.grid_type / _em_software.details / _em_software.name
Revision 1.3Oct 24, 2018Group: Advisory / Data collection / Derived calculations
Category: pdbx_validate_close_contact / struct_conn / struct_conn_type

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Structure visualization

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  • Deposited structure unit
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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
P: DNA Primer Strand
T: DNA Template Strand
F: DNA polymerase III subunit theta


Theoretical massNumber of molelcules
Total (without water)230,4327
Polymers230,4327
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area16940 Å2
ΔGint-84 kcal/mol
Surface area91740 Å2
MethodPISA

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Components

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DNA polymerase III subunit ... , 4 types, 5 molecules ABCDF

#1: Protein DNA polymerase III subunit alpha / DNA polymerase III holoenzyme


Mass: 103554.422 Da / Num. of mol.: 1 / Mutation: A921L, M923L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K12 (bacteria) / Gene: dnaE, polC, b0184, JW0179 / Production host: Escherichia coli BL21(DE3) (bacteria) / 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 K12 (bacteria) / Gene: dnaN, b3701, JW3678 / Production host: Escherichia coli BL21(DE3) (bacteria) / 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: T183L M185L A186P F187L
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K12 (bacteria) / Gene: dnaQ, mutD, b0215, JW0205 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03007, DNA-directed DNA polymerase
#6: Protein DNA polymerase III subunit theta / DNA polymerase III holoenzyme


Mass: 6503.385 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli K12 (bacteria) / Gene: holE, b1842, JW1831 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P0ABS8, DNA-directed DNA polymerase

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DNA chain , 2 types, 2 molecules PT

#4: DNA chain DNA Primer Strand


Mass: 5275.448 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: synthetic construct (others)
#5: DNA chain DNA Template Strand


Mass: 6718.339 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: 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

Component
IDNameTypeEntity IDParent-IDSourceDetails
1DNA polymerase III alpha, beta, epsilon, theta complex with mismatched DNA duplexCOMPLEXall0MULTIPLE SOURCES
2DNA polymerase III alpha, beta, epsilon, theta complex with mismatched DNA duplexCOMPLEX#1-#3, #61RECOMBINANTMap obtained after signal subtraction of the beta subunit and alignment of the remaining parts. Final reconstruction obtained with non-subtracted images and angles from local alignment
3mismatched DNA duplexCOMPLEX#4-#51RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.250 MDaNO
12
13
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
11Escherichia coli K12 (bacteria)83333K12
22Escherichia coli K12 (bacteria)83333K12
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
11Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)pET28a
22Escherichia coli BL21(DE3) (bacteria)469008BL21(DE3)pET28a
33synthetic construct (others)32630
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMHepes1
250 mMPotassium glutamate1
35 mMMagnesium Acetate1
42 mMDithiothreitol1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample was run over a gel filtration column prior to vitrification
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Prior to sample preparation 0.1 volumes of 0.05% Tween 20 were added to the sample 3 microliters were pipetted onto the grid and blotted for 4 seconds

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
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: 79545 X / Nominal defocus max: 3500 nm / Nominal defocus min: 1800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 K / Temperature (min): 80 K
Image recordingAverage exposure time: 25 sec. / Electron dose: 2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 1157
EM imaging opticsEnergyfilter name: GIF Quantum / Energyfilter upper: 20 eV / Energyfilter lower: 0 eV
Image scansWidth: 3710 / Height: 3710 / Movie frames/image: 20 / Used frames/image: 1-20

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Processing

EM software
IDNameVersionCategoryDetails
1RELION2particle selection
2DigitalMicrograph2.3image acquisitionManual collection
3Titan User Interfaceimage acquisitionLow dose
5GctfCTF correction
8Coot0.8.2model fitting
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
14REFMAC5.8model refinement
15LIBGmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 150000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 15616 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: OTHER
Details: The cryo-EM structure of the PolIIIalpha-clamp-exonuclease complex in the polymerase mode (PDB code: 5FKW) was used as a starting model, and the NMR structure of theta bound to the ...Details: The cryo-EM structure of the PolIIIalpha-clamp-exonuclease complex in the polymerase mode (PDB code: 5FKW) was used as a starting model, and the NMR structure of theta bound to the exonuclease catalytic domain (PDB code: 2XY8) was used to place theta into the cryo-EM map. The model was manually adjusted in Coot and geometry of the protein optimized in Refmac5 using DNA-specific restraints generated in LibG

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