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

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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 / DNA Binding protein
Function / homologyBacterial DNA polymerase III alpha subunit finger domain / DNA polymerase III-theta superfamily / Ribonuclease H-like superfamily / Exonuclease, RNase T/DNA polymerase III / Polymerase/histidinol phosphatase-like / 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, helix-hairpin-helix motif / Ribonuclease H superfamily ...Bacterial DNA polymerase III alpha subunit finger domain / DNA polymerase III-theta superfamily / Ribonuclease H-like superfamily / Exonuclease, RNase T/DNA polymerase III / Polymerase/histidinol phosphatase-like / 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, helix-hairpin-helix motif / Ribonuclease H superfamily / DNA polymerase III beta subunit, N-terminal domain / DNA polymerase 3, epsilon subunit / Exonuclease / OB-fold nucleic acid binding domain / DNA polymerase III beta subunit, central domain / DNA polymerase III beta subunit, C-terminal domain / PHP domain / DNA polymerase III, theta subunit / Bacterial DNA polymerase III alpha NTPase domain / Helix-hairpin-helix motif / DNA polymerase III-theta, bacterial / Bacterial DNA polymerase III, alpha subunit, NTPase domain / DNA polymerase III epsilon subunit, exonuclease domain / OB-fold nucleic acid binding domain, AA-tRNA synthetase-type / PHP domain / Polymerase/histidinol phosphatase, N-terminal / DNA polymerase III, beta sliding clamp / DNA polymerase III, alpha subunit / DNA polymerase III, core complex / DNA replication proofreading / DNA polymerase III complex / DNA strand elongation involved in DNA replication / nucleic acid phosphodiester bond hydrolysis / exonuclease activity / 3'-5' exonuclease activity / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / cellular response to DNA damage stimulus / DNA binding / identical protein binding / metal ion binding / cytosol / cytoplasm / DNA polymerase III subunit epsilon / Beta sliding clamp / DNA polymerase III subunit theta / DNA polymerase III subunit alpha
Function and homology information
Specimen sourceEscherichia coli K12 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 6.7 Å resolution
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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 10, 2016 / Release: Jan 18, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 18, 2017Structure modelrepositoryInitial release
1.1Feb 15, 2017Structure modelDatabase references
1.2Aug 30, 2017Structure modelData collection / Derived calculations / Experimental preparationem_imaging_optics / em_sample_support / em_software / struct_conn_em_imaging_optics.energyfilter_name / _em_sample_support.grid_type / _em_software.details / _em_software.name
1.3Oct 24, 2018Structure modelAdvisory / Data collection / Derived calculationspdbx_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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  • Superimposition on EM map
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Structure viewerMolecule:
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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
Polyers230,4327
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)16940
ΔGint (kcal/M)-84
Surface area (Å2)91740
MethodPISA

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Components

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

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


Mass: 103554.422 Da / Num. of mol.: 1 / Mutation: A921L, M923L / 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/peptide DNA polymerase III subunit beta / DNA polymerase III holoenzyme / Beta sliding clamp / Beta clamp


Mass: 40630.508 Da / Num. of mol.: 2 / 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/peptide DNA polymerase III subunit epsilon / DNA polymerase III holoenzyme


Mass: 27118.984 Da / Num. of mol.: 1 / Mutation: T183L M185L A186P F187L / 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/peptide DNA polymerase III subunit theta / DNA polymerase III holoenzyme


Mass: 6503.385 Da / Num. of mol.: 1 / 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: (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: (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 / Reconstruction method: single particle reconstruction

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Sample preparation

Component

Type: COMPLEX

IDNameEntity IDParent IDSourceDetails
1DNA polymerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex1, 2, 3, 4, 5, 60MULTIPLE SOURCES
2DNA polymerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex1, 2, 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 duplex4, 51RECOMBINANT
Molecular weight
IDValueEntity assembly IDExperimental value
10.250 MDa1NO
12
13
Source (natural)
IDEntity assembly IDNcbi tax IDOrganismStrain
1183333Escherichia coli K12 (bacteria)K12
2283333Escherichia coli K12 (bacteria)K12
3332630synthetic construct (others)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismPlasmidStrain
11469008Escherichia coli BL21(DE3) (bacteria)pET28aBL21(DE3)
22469008Escherichia coli BL21(DE3) (bacteria)pET28aBL21(DE3)
3332630synthetic construct (others)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer ID
120 mMHepes1
250 mMPotassium glutamate1
35 mMMagnesium Acetate1
42 mMDithiothreitol1
SpecimenConc.: 0.25
Details: Sample was run over a gel filtration column prior to vitrification
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277
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
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 64000 / Calibrated magnification: 79545 / Nominal defocus max: 3500 / Nominal defocus min: 1800 / Cs: 2.7 / C2 aperture diameter: 50 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 80 / Temperature (min): 80
Image recordingAverage exposure time: 25 / Electron dose: 2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 3 / Number of real images: 1157
EM imaging opticsEnergyfilter name: GIF Quantum / Energyfilter upper: 20 / Energyfilter lower: 0
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 selectionNumber of particles selected: 150000
SymmetryPoint symmetry: C1
3D reconstructionResolution: 6.7 / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 15616 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingDetails: 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
Ref protocol: OTHER

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