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- EMDB-10217: Cryo-EM structure of the RecBCD no Chi negative control complex -

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Basic information

Entry
Database: EMDB / ID: EMD-10217
TitleCryo-EM structure of the RecBCD no Chi negative control complex
Map data
SampleRecBCD bound to a forked DNA substrate that does not contain Chi:
RecBCD / DNA / (RecBCD enzyme subunit ...) x 3 / nucleic-acidNucleic acid
Function / homology
Function and homology information


exodeoxyribonuclease V activity / exodeoxyribonuclease V / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA translocase activity / single-stranded DNA helicase activity / recombinational repair / 3'-5' DNA helicase activity / ATPase, acting on DNA / endodeoxyribonuclease activity ...exodeoxyribonuclease V activity / exodeoxyribonuclease V / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA translocase activity / single-stranded DNA helicase activity / recombinational repair / 3'-5' DNA helicase activity / ATPase, acting on DNA / endodeoxyribonuclease activity / double-strand break repair via homologous recombination / helicase activity / response to radiation / DNA helicase activity / DNA recombination / DNA repair / cellular response to DNA damage stimulus / magnesium ion binding / DNA binding / ATP binding / cytosol
AAA+ ATPase domain / DNA helicase, UvrD/REP type / RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / Restriction endonuclease type II-like / Exonuclease, phage-type/RecB, C-terminal / DExx box DNA helicase domain superfamily / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase, C-terminal ...AAA+ ATPase domain / DNA helicase, UvrD/REP type / RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / Restriction endonuclease type II-like / Exonuclease, phage-type/RecB, C-terminal / DExx box DNA helicase domain superfamily / UvrD-like helicase, ATP-binding domain / UvrD-like DNA helicase, C-terminal / P-loop containing nucleoside triphosphate hydrolase / UvrD-like helicase C-terminal domain / UvrD/AddA helicase, N-terminal / PD-(D/E)XK endonuclease-like domain, AddAB-type / RecC, C-terminal / RecBCD enzyme subunit RecD, N-terminal domain
RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecBCD enzyme subunit RecB
Biological speciesEscherichia coli (E. coli) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsCheng K / Wilkinson M / Wigley DB
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.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionAug 13, 2019-
Header (metadata) releaseJan 1, 2020-
Map releaseJan 1, 2020-
UpdateJan 29, 2020-
Current statusJan 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6sjg
  • Surface level: 0.01
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10217.png

Downloads & links

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Map

FileDownload / File: emd_10217.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 240 pix.
= 251.52 Å		1.05 Å/pix.
x 240 pix.
= 251.52 Å		1.05 Å/pix.
x 240 pix.
= 251.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.048 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.01 / Movie #1: 0.01
Minimum - Maximum-0.023008011 - 0.05779179
Average (Standard dev.)0.00001524390 (±0.002595359)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 251.51999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0481.0481.048
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z251.520251.520251.520
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0230.0580.000

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Supplemental data

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Sample components

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Entire RecBCD bound to a forked DNA substrate that does not contain Chi

EntireName: RecBCD bound to a forked DNA substrate that does not contain Chi
Number of components: 7

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Component #1: protein, RecBCD bound to a forked DNA substrate that does not con...

ProteinName: RecBCD bound to a forked DNA substrate that does not contain Chi
Recombinant expression: No
MassTheoretical: 355 kDa

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Component #2: protein, RecBCD

ProteinName: RecBCD / Recombinant expression: No
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #3: protein, DNA

ProteinName: DNA / Recombinant expression: No
SourceSpecies: synthetic construct (others)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: protein, RecBCD enzyme subunit RecB

ProteinName: RecBCD enzyme subunit RecB / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 134.123688 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #5: protein, RecBCD enzyme subunit RecC

ProteinName: RecBCD enzyme subunit RecC / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 128.974102 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #6: protein, RecBCD enzyme subunit RecD

ProteinName: RecBCD enzyme subunit RecD / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 66.990367 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: nucleic-acid, Forked DNA substrate

nucleic acidName: Forked DNA substrate / Class: DNA
Details: We generated a forked DNA substrate with single-stranded overhangs by annealing two synthesised oligonucleotides
Structure: OTHER / Synthetic: No
Sequence: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DG)(DA)(DG)(DC)(DG) (DA)(DC)(DT)(DG)(DC)(DA)(DC)(DT)(DA)(DC) (DA)(DA)(DC)(DA)(DG)(DA)(DA)(DC)(DC)(DA) (DT)(DG)(DG)(DT)(DT)(DC) ...Sequence:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DG)(DA)(DG)(DC)(DG) (DA)(DC)(DT)(DG)(DC)(DA)(DC)(DT)(DA)(DC) (DA)(DA)(DC)(DA)(DG)(DA)(DA)(DC)(DC)(DA) (DT)(DG)(DG)(DT)(DT)(DC)(DT)(DG)(DT)(DT) (DG)(DT)(DA)(DG)(DT)(DG)(DC)(DA)(DG)(DT) (DC)(DG)(DC)(DT)(DC)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)
MassTheoretical: 26.049602 kDa
SourceSpecies: synthetic construct (others)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.2 mg/mL / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 90 % / Details: 1s blot before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 130000.0 X (nominal), 47710.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1400.0 - 2600.0 nm
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 788 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 62812
3D reconstructionSoftware: RELION / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Details: Rounds of manual structure editing in coot and real-space refinement with Phenix
Input PDB model: 6SJF

6sjf

Output model

6sjg

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