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- PDB-5ld2: Cryo-EM structure of RecBCD+DNA complex revealing activated nucle... -
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Open data
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Basic information
Entry | Database: PDB / ID: 5ld2 | ||||||
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Title | Cryo-EM structure of RecBCD+DNA complex revealing activated nuclease domain | ||||||
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![]() | HYDROLASE / Helicase / Nuclease / SH3 / Homologous Recombination | ||||||
Function / homology | ![]() exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA translocase activity / DNA 5'-3' helicase / single-stranded DNA helicase activity / DNA 3'-5' helicase / recombinational repair / 3'-5' DNA helicase activity ...exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA translocase activity / DNA 5'-3' helicase / single-stranded DNA helicase activity / DNA 3'-5' helicase / recombinational repair / 3'-5' DNA helicase activity / ATP-dependent activity, acting on DNA / DNA helicase activity / isomerase activity / DNA endonuclease activity / helicase activity / double-strand break repair via homologous recombination / response to radiation / 5'-3' DNA helicase activity / DNA recombination / DNA damage response / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / cytosol Similarity search - Function | ||||||
Biological species | ![]() ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.83 Å | ||||||
![]() | Wilkinson, M. / Chaban, Y. / Wigley, D.B. | ||||||
![]() | ![]() Title: Mechanism for nuclease regulation in RecBCD. Authors: Martin Wilkinson / Yuriy Chaban / Dale B Wigley / ![]() Abstract: In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly ...In bacterial cells, processing of double-stranded DNA breaks for repair by homologous recombination is catalysed by AddAB, AdnAB or RecBCD-type helicase-nucleases. These enzyme complexes are highly processive, duplex unwinding and degrading machines that require tight regulation. Here, we report the structure of E.coli RecBCD, determined by cryoEM at 3.8 Å resolution, with a DNA substrate that reveals how the nuclease activity of the complex is activated once unwinding progresses. Extension of the 5'-tail of the unwound duplex induces a large conformational change in the RecD subunit, that is transferred through the RecC subunit to activate the nuclease domain of the RecB subunit. The process involves a SH3 domain that binds to a region of the RecB subunit in a binding mode that is distinct from others observed previously in SH3 domains and, to our knowledge, this is the first example of peptide-binding of an SH3 domain in a bacterial system. | ||||||
History |
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 611.4 KB | Display | ![]() |
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PDB format | ![]() | 482.9 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 4038MC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
-RecBCD enzyme subunit ... , 3 types, 3 molecules BCD
#1: Protein | Mass: 133717.391 Da / Num. of mol.: 1 / Mutation: D1080A,D1080A,D1080A Source method: isolated from a genetically manipulated source Details: Residues 913-932 modelled as poly-Alanine in model to reflect uncertainty of the exact position of this loop due to weak density. Source: (gene. exp.) ![]() ![]() Strain: K12 / Gene: recB, ior, rorA, b2820, JW2788 / Plasmid: pETduet / Production host: ![]() ![]() |
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#2: Protein | Mass: 128974.102 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: K12 / Gene: recC, b2822, JW2790 / Plasmid: pRSFduet / Production host: ![]() ![]() |
#3: Protein | Mass: 67047.422 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() Strain: K12 / Gene: recD, hopE, b2819, JW2787 / Plasmid: pCDFduet / Production host: ![]() ![]() |
-DNA chain , 1 types, 1 molecules X
#4: DNA chain | Mass: 21440.707 Da / Num. of mol.: 1 / Source method: obtained synthetically Details: Hairpin=38-42 Duplex=13-37&43-67 5'Tail=1-12 3'Tail=68-70 Source: (synth.) ![]() |
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-Non-polymers , 2 types, 2 molecules ![](data/chem/img/ANP.gif)
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#5: Chemical | ChemComp-ANP / |
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#6: Chemical | ChemComp-MG / |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Ternary complex of RecBCD with forked DNA substrate and ADPNP Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Molecular weight | Value: 0.35 MDa / Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Specimen support | Details: Glow discharge was used to thin the carbon film, but allowed to dissipate prior to use. Detergent or amphipols treatment was used to render carbon hydrophilic. See article for details. Grid type: C-flat | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Calibrated magnification: 37313 X / Calibrated defocus min: 500 nm / Calibrated defocus max: 3800 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 0.4 sec. / Electron dose: 1.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1674 |
EM imaging optics | Energyfilter name: GIF |
Image scans | Sampling size: 5 µm / Movie frames/image: 30 / Used frames/image: 1-30 |
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Processing
Software | Name: PHENIX / Version: 1.10.1_2155: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74656 / Symmetry type: POINT |