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- EMDB-4142: Cryo-EM structure of the E. coli replicative DNA polymerase-clamp... -

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Basic information

Entry
Database: EMDB / ID: EMD-4142
TitleCryo-EM structure of the E. coli replicative DNA polymerase-clamp-exonuclase-theta complex bound to DNA in the editing mode
Map dataRelated to EMD-4142 Local alignment after signal subtraction of the beta subunit
Sample
  • Complex: DNA polyerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex
    • Complex: DNA polyerase III alpha, epsilon, theta complex with mismatched DNA duplex
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 polymerase III subunit epsilon / Beta sliding clamp / DNA polymerase III subunit theta / DNA polymerase III subunit alpha
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.7 Å
AuthorsFernandez-Leiro R / Conrad J / Scheres SHW / Lamers MH
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 12, 2016-
Header (metadata) releaseOct 26, 2016-
Map releaseJan 18, 2017-
UpdateAug 30, 2017-
Current statusAug 30, 2017Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4142.png

Downloads & links

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Map

FileDownload / File: emd_4142.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRelated to EMD-4142 Local alignment after signal subtraction of the beta subunit
Voxel sizeX=Y=Z: 1.76 Å
Density
Contour LevelBy AUTHOR: 0.08 / Movie #1: 0.08
Minimum - Maximum-0.14866291 - 0.33609918
Average (Standard dev.)0.00007027035 (±0.016236395)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 246.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.761.761.76
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z246.400246.400246.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-190
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.1490.3360.000

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Supplemental data

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Mask #1

Fileemd_4142_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_4142_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_4142_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : DNA polyerase III alpha, beta, epsilon, theta complex with mismat...

EntireName: DNA polyerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex
Components
  • Complex: DNA polyerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex
    • Complex: DNA polyerase III alpha, epsilon, theta complex with mismatched DNA duplex

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Supramolecule #1: DNA polyerase III alpha, beta, epsilon, theta complex with mismat...

SupramoleculeName: DNA polyerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) / Recombinant plasmid: pET28a
Molecular weightTheoretical: 250 KDa

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Supramolecule #2: DNA polyerase III alpha, epsilon, theta complex with mismatched D...

SupramoleculeName: DNA polyerase III alpha, epsilon, theta complex with mismatched DNA duplex
type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1, #3-#6
Details: Map 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
Source (natural)Organism: Escherichia coli (E. coli) / Strain: K12
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) / Recombinant plasmid: pET28a

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.25 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHepes
50.0 mMPotassium glutamate
5.0 mMMagnesium Acetate
2.0 mMDithiothreitol
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
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.
DetailsSample was run over a gel filtration column prior to vitrification

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 79545 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 64000
Specialist opticsEnergy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 80.0 K / Max: 80.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3710 pixel / Number grids imaged: 3 / Number real images: 1157 / Average exposure time: 25.0 sec. / Average electron dose: 2.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 150000
CTF correctionSoftware - Name: Gctf
Startup modelType of model: EMDB MAP
EMDB ID:

3198


Details: low pass filtered to 60 angtrom
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2)
Final 3D classificationSoftware - Name: RELION (ver. 2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2)
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2) / Number images used: 15616
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsThe cryo-EM structure of the PolIIIalpha-clamp-exonuclease complex in the polymerase mode (PDB code: 5FKW)1 was used as a starting model, and the NMR structure of theta bound to the ? catalytic domain (PDB code: 2XY8)13 was used to place ? into the cryo-EM map. The model was manually adjusted in Coot35 and geometry of the protein optimized in Refmac536 using DNA-specific restraints generated in LibG36
RefinementProtocol: OTHER

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