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Yorodumi- EMDB-10401: Cryo-EM structure of the DNA-bound PolD-PCNA processive complex f... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-10401 | ||||||||||||
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Title | Cryo-EM structure of the DNA-bound PolD-PCNA processive complex from P. abyssi | ||||||||||||
Map data | Post processed and masked map of the PolD-PCNA-DNA from P. abyssi | ||||||||||||
Sample |
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Function / homology | Function and homology information exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intein-mediated protein splicing / DNA catabolic process / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / leading strand elongation / regulation of DNA replication / mismatch repair / translesion synthesis ...exodeoxyribonuclease I / single-stranded DNA 3'-5' DNA exonuclease activity / intein-mediated protein splicing / DNA catabolic process / DNA strand elongation involved in DNA replication / DNA polymerase processivity factor activity / leading strand elongation / regulation of DNA replication / mismatch repair / translesion synthesis / DNA-templated DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding Similarity search - Function | ||||||||||||
Biological species | Pyrococcus abyssi (archaea) / synthetic construct (others) / Pyrococcus abyssi (strain GE5 / Orsay) (archaea) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.77 Å | ||||||||||||
Authors | Madru C / Raia P / Hugonneau Beaufet I / Pehau-Arnaudet G / England P / Lindhal E / Delarue M / Carroni M / Sauguet L | ||||||||||||
Funding support | France, Sweden, 3 items
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Citation | Journal: Nat Commun / Year: 2020 Title: Structural basis for the increased processivity of D-family DNA polymerases in complex with PCNA. Authors: Clément Madru / Ghislaine Henneke / Pierre Raia / Inès Hugonneau-Beaufet / Gérard Pehau-Arnaudet / Patrick England / Erik Lindahl / Marc Delarue / Marta Carroni / Ludovic Sauguet / Abstract: Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced ...Replicative DNA polymerases (DNAPs) have evolved the ability to copy the genome with high processivity and fidelity. In Eukarya and Archaea, the processivity of replicative DNAPs is greatly enhanced by its binding to the proliferative cell nuclear antigen (PCNA) that encircles the DNA. We determined the cryo-EM structure of the DNA-bound PolD-PCNA complex from Pyrococcus abyssi at 3.77 Å. Using an integrative structural biology approach - combining cryo-EM, X-ray crystallography, protein-protein interaction measurements, and activity assays - we describe the molecular basis for the interaction and cooperativity between a replicative DNAP and PCNA. PolD recruits PCNA via a complex mechanism, which requires two different PIP-boxes. We infer that the second PIP-box, which is shared with the eukaryotic Polα replicative DNAP, plays a dual role in binding either PCNA or primase, and could be a master switch between an initiation and a processive phase during replication. | ||||||||||||
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_10401.map.gz | 9.9 MB | EMDB map data format | |
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Header (meta data) | emd-10401-v30.xml emd-10401.xml | 29.6 KB 29.6 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_10401_fsc.xml | 10.8 KB | Display | FSC data file |
Images | emd_10401.png | 129.7 KB | ||
Masks | emd_10401_msk_1.map | 103 MB | Mask map | |
Others | emd_10401_additional_1.map.gz emd_10401_additional_2.map.gz emd_10401_half_map_1.map.gz emd_10401_half_map_2.map.gz | 96.4 MB 80.5 MB 80.9 MB 80.9 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-10401 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10401 | HTTPS FTP |
-Validation report
Summary document | emd_10401_validation.pdf.gz | 408.5 KB | Display | EMDB validaton report |
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Full document | emd_10401_full_validation.pdf.gz | 407.6 KB | Display | |
Data in XML | emd_10401_validation.xml.gz | 16.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10401 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10401 | HTTPS FTP |
-Related structure data
Related structure data | 6t8hMC 6t7xC 6t7yC M: atomic model generated by this map C: citing same article (ref.) |
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Similar 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_10401.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Post processed and masked map of the PolD-PCNA-DNA from P. abyssi | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.83 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_10401_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Post processed unmasked map of the PolD-PCNA-DNA from P. abyssi
File | emd_10401_additional_1.map | ||||||||||||
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Annotation | Post processed unmasked map of the PolD-PCNA-DNA from P. abyssi | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Unfiltered map of the PolD-PCNA-DNA from P. abyssi
File | emd_10401_additional_2.map | ||||||||||||
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Annotation | Unfiltered map of the PolD-PCNA-DNA from P. abyssi | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2 of the PolD-PCNA-DNA from P. abyssi
File | emd_10401_half_map_1.map | ||||||||||||
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Annotation | Half map 2 of the PolD-PCNA-DNA from P. abyssi | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 1 of the PolD-PCNA-DNA from P. abyssi
File | emd_10401_half_map_2.map | ||||||||||||
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Annotation | Half map 1 of the PolD-PCNA-DNA from P. abyssi | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
+Entire : DNA-bound PolD-PCNA processive complex from P. abyssi
+Supramolecule #1: DNA-bound PolD-PCNA processive complex from P. abyssi
+Supramolecule #2: PolD-PCNA processive complex
+Supramolecule #3: DNA
+Macromolecule #1: DNA polymerase sliding clamp
+Macromolecule #2: DNA polymerase II small subunit
+Macromolecule #3: DP2 subunit of D-family DNA-polymerase
+Macromolecule #4: DNA primer
+Macromolecule #5: DNA template
+Macromolecule #6: FE (III) ION
+Macromolecule #7: ZINC ION
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.1 mg/mL |
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Buffer | pH: 8 |
Grid | Model: C-flat-2/2 4C / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: 5mA current |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 4602 / Average exposure time: 5.0 sec. / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated defocus max: 3.5 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus min: 0.5 µm |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |