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- PDB-5ld2: Cryo-EM structure of RecBCD+DNA complex revealing activated nucle... -

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Basic information

Entry
Database: PDB / ID: 5ld2
TitleCryo-EM structure of RecBCD+DNA complex revealing activated nuclease domain
Components
  • (RecBCD enzyme subunit ...) x 3
  • Fork-Hairpin DNA (70-MER)
KeywordsHYDROLASE / Helicase / Nuclease / SH3 / Homologous Recombination
Function / homology
Function and homology information


exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA translocase activity / single-stranded DNA helicase activity / recombinational repair / 3'-5' DNA helicase activity / ATP-dependent activity, acting on DNA / DNA helicase activity ...exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA translocase activity / single-stranded DNA helicase activity / recombinational repair / 3'-5' DNA helicase activity / ATP-dependent activity, acting on DNA / DNA helicase activity / DNA endonuclease activity / helicase activity / double-strand break repair via homologous recombination / response to radiation / DNA recombination / DNA damage response / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / cytosol
Similarity search - Function
RecBCD complex, subunit RecD, N-terminal domain / Arc Repressor Mutant, subunit A - #990 / Recbcd, chain B, domain 2 / Recbcd, chain B, domain 2 / Rossmann fold - #10930 / Arc Repressor Mutant, subunit A - #160 / Lambda Exonuclease; Chain A - #10 / PCRA; domain 4 / PCRA; domain 4 / RecBCD enzyme subunit RecB ...RecBCD complex, subunit RecD, N-terminal domain / Arc Repressor Mutant, subunit A - #990 / Recbcd, chain B, domain 2 / Recbcd, chain B, domain 2 / Rossmann fold - #10930 / Arc Repressor Mutant, subunit A - #160 / Lambda Exonuclease; Chain A - #10 / PCRA; domain 4 / PCRA; domain 4 / RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecC, C-terminal / RecBCD enzyme subunit RecD, N-terminal domain / Exodeoxyribonuclease V, gamma subunit / RecC C-terminal domain / RecBCD enzyme subunit RecD, N-terminal domain / Lambda Exonuclease; Chain A / PD-(D/E)XK endonuclease-like domain, AddAB-type / PD-(D/E)XK nuclease superfamily / DExx box DNA helicase domain superfamily / AAA domain / UvrD-like DNA helicase C-terminal domain profile. / UvrD-like DNA helicase, C-terminal / UvrD-like helicase C-terminal domain / UvrD-like helicase C-terminal domain / UvrD-like helicase C-terminal domain / UvrD/REP helicase N-terminal domain / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / PD-(D/E)XK endonuclease-like domain superfamily / AAA domain / Restriction endonuclease type II-like / Arc Repressor Mutant, subunit A / P-loop containing nucleotide triphosphate hydrolases / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / Alpha-Beta Complex / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / Orthogonal Bundle / 3-Layer(aba) Sandwich / Mainly Alpha / Alpha Beta
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecBCD enzyme subunit RecB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Endothia gyrosa (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.83 Å
AuthorsWilkinson, M. / Chaban, Y. / Wigley, D.B.
CitationJournal: Elife / Year: 2016
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
DepositionJun 23, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 5, 2016Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Data collection / Derived calculations / Experimental preparation
Category: em_sample_support / em_software / struct_conn
Item: _em_sample_support.grid_type / _em_software.details / _em_software.name
Revision 1.2Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
B: RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB
C: RecBCD enzyme subunit RecC
D: RecBCD enzyme subunit RecD
X: Fork-Hairpin DNA (70-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)351,7106
Polymers351,1804
Non-polymers5312
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area24420 Å2
ΔGint-114 kcal/mol
Surface area129230 Å2
MethodPISA

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Components

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RecBCD enzyme subunit ... , 3 types, 3 molecules BCD

#1: Protein RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB / Exodeoxyribonuclease V 135 kDa polypeptide / Exodeoxyribonuclease V beta chain / Exonuclease V ...Exodeoxyribonuclease V 135 kDa polypeptide / Exodeoxyribonuclease V beta chain / Exonuclease V subunit RecB / ExoV subunit RecB / Helicase/nuclease RecBCD subunit RecB


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.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: recB, ior, rorA, b2820, JW2788 / Plasmid: pETduet / Production host: Escherichia coli (E. coli) / Strain (production host): B834 / References: UniProt: P08394, exodeoxyribonuclease V
#2: Protein RecBCD enzyme subunit RecC / Exodeoxyribonuclease V 125 kDa polypeptide / Exodeoxyribonuclease V gamma chain / Exonuclease V ...Exodeoxyribonuclease V 125 kDa polypeptide / Exodeoxyribonuclease V gamma chain / Exonuclease V subunit RecC / ExoV subunit RecC


Mass: 128974.102 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: recC, b2822, JW2790 / Plasmid: pRSFduet / Production host: Escherichia coli (E. coli) / Strain (production host): B834 / References: UniProt: P07648, exodeoxyribonuclease V
#3: Protein RecBCD enzyme subunit RecD / Exodeoxyribonuclease V 67 kDa polypeptide / Exodeoxyribonuclease V alpha chain / Exonuclease V ...Exodeoxyribonuclease V 67 kDa polypeptide / Exodeoxyribonuclease V alpha chain / Exonuclease V subunit RecD / ExoV subunit RecD / Helicase/nuclease RecBCD subunit RecD


Mass: 67047.422 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (strain K12) (bacteria)
Strain: K12 / Gene: recD, hopE, b2819, JW2787 / Plasmid: pCDFduet / Production host: Escherichia coli (E. coli) / Strain (production host): B834 / References: UniProt: P04993, exodeoxyribonuclease V

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DNA chain , 1 types, 1 molecules X

#4: DNA chain Fork-Hairpin DNA (70-MER)


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.) Endothia gyrosa (fungus)

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#6: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Ternary complex of RecBCD with forked DNA substrate and ADPNP
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.35 MDa / Experimental value: NO
Source (natural)Organism: Escherichia coli K12 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: B834 / Plasmid: 3 plasmids
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-HClTris1
250 mMSodium ChlorideNaClSodium chloride1
31 mMTCEP1
44 mMMagnesium ChlorideMgCl21
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 37313 X / Calibrated defocus min: 500 nm / Calibrated defocus max: 3800 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 opticsEnergyfilter name: GIF
Image scansSampling size: 5 µm / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION1.3particle selection
2EPUimage acquisition
4CTFFIND4CTF correction
5Gctf0.5CTF correctionUsed at the end to apply local CTF corrections for individual particles
8UCSF Chimeramodel fitting
9Cootmodel fitting
11REFMACmodel refinement
12PHENIXmodel refinementPhenix_real_space_refine
13RELION1.3initial Euler assignment
14RELION1.4final Euler assignment
16RELION1.43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.83 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74656 / Symmetry type: POINT

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