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Yorodumi- EMDB-4142: Cryo-EM structure of the E. coli replicative DNA polymerase-clamp... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-4142 | |||||||||
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Title | Cryo-EM structure of the E. coli replicative DNA polymerase-clamp-exonuclase-theta complex bound to DNA in the editing mode | |||||||||
Map data | Related to EMD-4142 Local alignment after signal subtraction of the beta subunit | |||||||||
Sample |
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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 / lagging strand elongation / replisome / exonuclease activity / regulation of DNA-templated DNA replication initiation / DNA replication proofreading ...DNA polymerase III, core complex / Hda-beta clamp complex / bacterial-type DNA replication / replication inhibiting complex / DNA polymerase III complex / lagging strand elongation / replisome / exonuclease activity / regulation of DNA-templated DNA replication initiation / DNA replication proofreading / DNA strand elongation involved in DNA replication / 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 / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 6.7 Å | |||||||||
Authors | Fernandez-Leiro R / Conrad J / Scheres SHW / Lamers MH | |||||||||
Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_4142.map.gz | 9.6 MB | EMDB map data format | |
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Header (meta data) | emd-4142-v30.xml emd-4142.xml | 20.8 KB 20.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_4142_fsc.xml | 5 KB | Display | FSC data file |
Images | emd_4142.png | 116.4 KB | ||
Masks | emd_4142_msk_1.map | 10.5 MB | Mask map | |
Others | emd_4142_half_map_1.map.gz emd_4142_half_map_2.map.gz | 7.9 MB 7.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-4142 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-4142 | HTTPS FTP |
-Validation report
Summary document | emd_4142_validation.pdf.gz | 465.9 KB | Display | EMDB validaton report |
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Full document | emd_4142_full_validation.pdf.gz | 465.1 KB | Display | |
Data in XML | emd_4142_validation.xml.gz | 10.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4142 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-4142 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_4142.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Related to EMD-4142 Local alignment after signal subtraction of the beta subunit | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.76 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_4142_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_4142_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_4142_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : DNA polyerase III alpha, beta, epsilon, theta complex with mismat...
Entire | Name: DNA polyerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex |
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Components |
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-Supramolecule #1: DNA polyerase III alpha, beta, epsilon, theta complex with mismat...
Supramolecule | Name: DNA polyerase III alpha, beta, epsilon, theta complex with mismatched DNA duplex type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#6 |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: K12 |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) / Recombinant plasmid: pET28a |
Molecular weight | Theoretical: 250 KDa |
-Supramolecule #2: DNA polyerase III alpha, epsilon, theta complex with mismatched D...
Supramolecule | Name: 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 |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: K12 |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: BL21 (DE3) / Recombinant plasmid: pET28a |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.25 mg/mL | ||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR | ||||||||||
Vitrification | Cryogen 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. | ||||||||||
Details | Sample was run over a gel filtration column prior to vitrification |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Temperature | Min: 80.0 K / Max: 80.0 K |
Specialist optics | Energy filter - Name: GIF Quantum / Energy filter - Lower energy threshold: 0 eV / Energy filter - Upper energy threshold: 20 eV |
Image recording | Film 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 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Calibrated magnification: 79545 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.8 µm / Nominal magnification: 64000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Details | The 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 |
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Refinement | Protocol: OTHER |