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- EMDB-3460: Cryo-EM structure of Lambda Phage protein GamS bound to RecBCD. -

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Basic information

Entry
Database: EMDB / ID: EMD-3460
TitleCryo-EM structure of Lambda Phage protein GamS bound to RecBCD.
Map data
SampleRecBCD complex bound to inhibitory protein lambda GamS:
RecBCD helicase/nuclease complex / Lambda GamS / (RecBCD enzyme subunit ...) x 3 / Host-nuclease inhibitor protein gam
Function / homology
Function and homology information


DNA end degradation evasion by virus / deoxyribonuclease inhibitor 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 ...DNA end degradation evasion by virus / deoxyribonuclease inhibitor 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 / 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
DNA helicase, UvrD/REP type / DExx box DNA helicase domain superfamily / AAA+ ATPase domain / RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / Host-nuclease inhibitor Gam / Restriction endonuclease type II-like / Exonuclease, phage-type/RecB, C-terminal / UvrD-like helicase, ATP-binding domain ...DNA helicase, UvrD/REP type / DExx box DNA helicase domain superfamily / AAA+ ATPase domain / RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / Host-nuclease inhibitor Gam / Restriction endonuclease type II-like / Exonuclease, phage-type/RecB, C-terminal / 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 / Host-nuclease inhibitor Gam superfamily
Host-nuclease inhibitor protein gam / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecBCD enzyme subunit RecB
Biological speciesEscherichia coli (E. coli) / Enterobacteria phage lambda (bacteriophage)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWilkinson M / Chaban Y / Wigley DB
CitationJournal: Elife / Year: 2016
Title: Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones.
Authors: Martin Wilkinson / Lucy Troman / Wan Ak Wan Nur Ismah / Yuriy Chaban / Matthew B Avison / Mark S Dillingham / Dale B Wigley /
Abstract: Our previous paper (Wilkinson , 2016) used high-resolution cryo-electron microscopy to solve the structure of the RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the ...Our previous paper (Wilkinson , 2016) used high-resolution cryo-electron microscopy to solve the structure of the RecBCD complex, which acts in both the repair of double-stranded DNA breaks and the degradation of bacteriophage DNA. To counteract the latter activity, bacteriophage λ encodes a small protein inhibitor called Gam that binds to RecBCD and inactivates the complex. Here, we show that Gam inhibits RecBCD by competing at the DNA-binding site. The interaction surface is extensive and involves molecular mimicry of the DNA substrate. We also show that expression of Gam in or increases sensitivity to fluoroquinolones; antibacterials that kill cells by inhibiting topoisomerases and inducing double-stranded DNA breaks. Furthermore, fluoroquinolone-resistance in clinical isolates is reversed by expression of Gam. Together, our data explain the synthetic lethality observed between topoisomerase-induced DNA breaks and the RecBCD gene products, suggesting a new co-antibacterial strategy.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionNov 8, 2016-
Header (metadata) releaseNov 16, 2016-
Map releaseJan 11, 2017-
UpdateOct 23, 2019-
Current statusOct 23, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5mbv
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3460.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 180 pix.
= 241.2 Å
1.34 Å/pix.
x 180 pix.
= 241.2 Å
1.34 Å/pix.
x 180 pix.
= 241.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.035 / Movie #1: 0.035
Minimum - Maximum-0.042780045 - 0.16285071
Average (Standard dev.)-0.0006984152 (±0.009656814)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 241.20001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z241.200241.200241.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.0430.163-0.001

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Supplemental data

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Sample components

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Entire RecBCD complex bound to inhibitory protein lambda GamS

EntireName: RecBCD complex bound to inhibitory protein lambda GamS
Details: RecBCD and GamS were expressed and purified separately. They were mixed with an excess of GamS then passed through a size exclusion column to separate out free GamS.
Number of components: 7

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Component #1: protein, RecBCD complex bound to inhibitory protein lambda GamS

ProteinName: RecBCD complex bound to inhibitory protein lambda GamS
Details: RecBCD and GamS were expressed and purified separately. They were mixed with an excess of GamS then passed through a size exclusion column to separate out free GamS.
Recombinant expression: No
MassTheoretical: 350 kDa

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Component #2: protein, RecBCD helicase/nuclease complex

ProteinName: RecBCD helicase/nuclease complex / Recombinant expression: No
MassTheoretical: 330 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: Multiple / Strain: BL21(DE3)

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Component #3: protein, Lambda GamS

ProteinName: Lambda GamS / Recombinant expression: No
MassTheoretical: 20 kDa
SourceSpecies: Enterobacteria phage lambda (bacteriophage)
Source (engineered)Expression System: Escherichia coli (E. coli) / Vector: pET22b / Strain: BL21(DE3)

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Component #4: protein, RecBCD enzyme subunit RecB

ProteinName: RecBCD enzyme subunit RecB / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 134.167703 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: 67.047422 kDa
SourceSpecies: Escherichia coli (E. coli)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Component #7: protein, Host-nuclease inhibitor protein gam

ProteinName: Host-nuclease inhibitor protein gam
Details: GamL is cleaved at position Y44 when expressed in E.coli into GamS (see paper - pubmed id 17544443). We had the GamS gene synthesised to start at M41 for this project. It purifies as a dimer.
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 11.661924 kDa
SourceSpecies: Enterobacteria phage lambda (bacteriophage)
Source (engineered)Expression System: Escherichia coli BL21(DE3) (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.4 mg/mL / pH: 7.5
Support filmGrids were thinned by glow discharge in 30s steps with 1 minute wait in between treatments. Then left for 1-2 weeks prior to overnight treatment with 1 mM Ampiphol A8-35 to render surface hydrophilic. Grids were washed with 5 drops of water prior to use. EMS
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 90 % / Details: 3 ul sample applied Blot force of -4 for 1 s.

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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: 1.4 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 105000.0 X (nominal), 37313.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200.0 - 2400.0 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: 334 / Sampling size: 5 µm

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 122796
Details: Frames were aligned using motioncorr prior to processing.
3D reconstructionSoftware: RELION / CTF correction: CTF correction done at start of processing / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement protocol: rigid body / Target criteria: Cross-correlation coefficient
Input PDB model: 5LD2
Modeling #2Refinement protocol: rigid body / Target criteria: Cross-correlation coefficient
Input PDB model: 2UUZ
Output model

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