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- PDB-6t2v: Cryo-EM structure of the RecBCD in complex with Chi-plus2 substrate -

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Basic information

Entry
Database: PDB / ID: 6t2v
TitleCryo-EM structure of the RecBCD in complex with Chi-plus2 substrate
Components
  • DNA (Chi-plus2)
  • RecBCD enzyme subunit RecB
  • RecBCD enzyme subunit RecC
  • RecBCD enzyme subunit RecD
KeywordsHYDROLASE / DNA repair / Homologous recombination / ATP hydrolysis / Helicase / Nuclease / translocation
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 / helicase activity / DNA endonuclease 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
DNA / DNA (> 10) / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecBCD enzyme subunit RecB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsCheng, K. / Wilkinson, M. / Wigley, D.B.
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: A conformational switch in response to Chi converts RecBCD from phage destruction to DNA repair.
Authors: Kaiying Cheng / Martin Wilkinson / Yuriy Chaban / Dale B Wigley /
Abstract: The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We ...The RecBCD complex plays key roles in phage DNA degradation, CRISPR array acquisition (adaptation) and host DNA repair. The switch between these roles is regulated by a DNA sequence called Chi. We report cryo-EM structures of the Escherichia coli RecBCD complex bound to several different DNA forks containing a Chi sequence, including one in which Chi is recognized and others in which it is not. The Chi-recognized structure shows conformational changes in regions of the protein that contact Chi and reveals a tortuous path taken by the DNA. Sequence specificity arises from interactions with both the RecC subunit and the sequence itself. These structures provide molecular details for how Chi is recognized and insights into the changes that occur in response to Chi binding that switch RecBCD from bacteriophage destruction and CRISPR spacer acquisition to constructive host DNA repair.
History
DepositionOct 9, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 1, 2020Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
B: RecBCD enzyme subunit RecB
C: RecBCD enzyme subunit RecC
D: RecBCD enzyme subunit RecD
X: DNA (Chi-plus2)


Theoretical massNumber of molelcules
Total (without water)356,8564
Polymers356,8564
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area23030 Å2
ΔGint-113 kcal/mol
Surface area131140 Å2
MethodPISA

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Components

#1: Protein 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: 134123.688 Da / Num. of mol.: 1 / Mutation: D1080A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: recB, ior, rorA, b2820, JW2788 / Production host: Escherichia coli (E. coli) / 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 (E. coli) / Gene: recC, b2822, JW2790 / Production host: Escherichia coli (E. coli) / 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: 66990.367 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: recD, hopE, b2819, JW2787 / Production host: Escherichia coli (E. coli) / References: UniProt: P04993, exodeoxyribonuclease V
#4: DNA chain DNA (Chi-plus2)


Mass: 26768.045 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: We generated a forked DNA substrate with single-stranded overhangs by annealing two synthesised oligonucleotides
Source: (synth.) synthetic construct (others)

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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

Component
IDNameTypeEntity IDParent-IDSource
1RecBCD bound to a forked DNA substrate that contain Chi-plus2COMPLEXall0MULTIPLE SOURCES
2RecBdCDCOMPLEX#1-#31RECOMBINANT
3Chi-plus2COMPLEX#41RECOMBINANT
Molecular weightValue: 0.355 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Escherichia coli (E. coli)562
23synthetic construct (others)32630
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTRIS-HClTris1
250 mMNaClSodium chloride1
30.5 mMTCEP1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K / Details: 1s blot before plunging

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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 / Nominal defocus max: 2700 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1 sec. / Electron dose: 75.9 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3613
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

Software
NameVersionClassificationNB
PHENIX1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2EPUimage acquisition
4GctfCTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.12model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 754150
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 380912 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: Rounds of manual structure editing in coot and real-space refinement with Phenix
Atomic model buildingPDB-ID: 5LD2
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 237.94 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001424452
ELECTRON MICROSCOPYf_angle_d0.396633409
ELECTRON MICROSCOPYf_chiral_restr0.03433673
ELECTRON MICROSCOPYf_plane_restr0.00524205
ELECTRON MICROSCOPYf_dihedral_angle_d16.683914583

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