[English] 日本語
Yorodumi- PDB-6t8h: Cryo-EM structure of the DNA-bound PolD-PCNA processive complex f... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6t8h | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of the DNA-bound PolD-PCNA processive complex from P. abyssi | ||||||||||||
Components |
| ||||||||||||
Keywords | REPLICATION / PCNA / DP2 / PolD / DP1 / PIP-box | ||||||||||||
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 / regulation of DNA replication / DNA-templated DNA replication / DNA replication / DNA-directed DNA polymerase ...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 / regulation of DNA replication / DNA-templated DNA replication / DNA replication / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / DNA binding Similarity search - Function | ||||||||||||
Biological species | Pyrococcus abyssi (archaea) synthetic construct (others) | ||||||||||||
Method | ELECTRON MICROSCOPY / 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, 3items
| ||||||||||||
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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6t8h.cif.gz | 446.2 KB | Display | PDBx/mmCIF format |
---|---|---|---|---|
PDB format | pdb6t8h.ent.gz | 359.7 KB | Display | PDB format |
PDBx/mmJSON format | 6t8h.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6t8h_validation.pdf.gz | 1.1 MB | Display | wwPDB validaton report |
---|---|---|---|---|
Full document | 6t8h_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | 6t8h_validation.xml.gz | 92.7 KB | Display | |
Data in CIF | 6t8h_validation.cif.gz | 135.1 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/t8/6t8h ftp://data.pdbj.org/pub/pdb/validation_reports/t8/6t8h | HTTPS FTP |
-Related structure data
Related structure data | 10401MC 6t7xC 6t7yC M: map data used to model this data C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
-Assembly
Deposited unit |
|
---|---|
1 |
|
-Components
-DNA polymerase ... , 2 types, 4 molecules ECDA
#1: Protein | Mass: 29471.764 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pyrococcus abyssi (strain GE5 / Orsay) (archaea) Strain: GE5 / Orsay / Gene: pcn, PYRAB13790, PAB1465 / Production host: Escherichia coli (E. coli) / References: UniProt: Q9UYX8 #2: Protein | | Mass: 69408.008 Da / Num. of mol.: 1 / Mutation: H451A Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pyrococcus abyssi (strain GE5 / Orsay) (archaea) Strain: GE5 / Orsay / Gene: polB, PYRAB01210, PAB2266 / Production host: Escherichia coli (E. coli) References: UniProt: Q9V2F3, DNA-directed DNA polymerase, exodeoxyribonuclease I |
---|
-Protein , 1 types, 1 molecules B
#3: Protein | Mass: 144762.188 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Pyrococcus abyssi (archaea) / Production host: Escherichia coli (E. coli) References: UniProt: Q9V2F4*PLUS, DNA-directed DNA polymerase |
---|
-DNA chain , 2 types, 2 molecules PT
#4: DNA chain | Mass: 5519.542 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
---|---|
#5: DNA chain | Mass: 7717.934 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) |
-Non-polymers , 2 types, 5 molecules
#6: Chemical | ChemComp-FE / |
---|---|
#7: Chemical | ChemComp-ZN / |
-Details
Has ligand of interest | N |
---|
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
---|---|
EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component |
| ||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Molecular weight | Value: 310 kDa/nm / Experimental value: YES | ||||||||||||||||||||||||
Source (natural) |
| ||||||||||||||||||||||||
Source (recombinant) |
| ||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||
Specimen | Conc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Details: 5mA current / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2 4C | ||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
---|---|
Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus min: 500 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 5 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4602 |
EM imaging optics | Energyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 40 / Used frames/image: 1-40 |
-Processing
EM software |
| ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||
Particle selection | Details: Indicated in the M&M | ||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||
3D reconstruction | Resolution: 3.77 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149200 / Symmetry type: POINT |