+Open data
-Basic information
Entry | Database: PDB / ID: 5mbv | ||||||
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Title | Cryo-EM structure of Lambda Phage protein GamS bound to RecBCD. | ||||||
Components |
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Keywords | DNA BINDING PROTEIN / Inhibitor / Complex / DNA repair / Helicase/Nuclease | ||||||
Function / homology | Function and homology information symbiont-mediated evasion of DNA end degradation by host / deoxyribonuclease inhibitor 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 ...symbiont-mediated evasion of DNA end degradation by host / deoxyribonuclease inhibitor 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 / symbiont-mediated suppression of host innate immune response / virus-mediated perturbation of host defense response / DNA damage response / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / cytosol Similarity search - Function | ||||||
Biological species | Escherichia coli (E. coli) Enterobacteria phage lambda (virus) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | ||||||
Authors | Wilkinson, M. / Chaban, Y. / Wigley, D.B. | ||||||
Citation | Journal: Elife / Year: 2016 Title: Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones. Authors: Martin Wilkinson / Luca 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. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 5mbv.cif.gz | 608.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb5mbv.ent.gz | 487.4 KB | Display | PDB format |
PDBx/mmJSON format | 5mbv.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 5mbv_validation.pdf.gz | 861.1 KB | Display | wwPDB validaton report |
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Full document | 5mbv_full_validation.pdf.gz | 889.1 KB | Display | |
Data in XML | 5mbv_validation.xml.gz | 76.8 KB | Display | |
Data in CIF | 5mbv_validation.cif.gz | 116.3 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/mb/5mbv ftp://data.pdbj.org/pub/pdb/validation_reports/mb/5mbv | HTTPS FTP |
-Related structure data
Related structure data | 3460MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 134167.703 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Gene: recB, ior, rorA, b2820, JW2788 / Plasmid: pETduet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P08394, exodeoxyribonuclease V |
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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.) Escherichia coli (E. coli) / Gene: recC, b2822, JW2790 / Plasmid: pRSFduet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P07648, exodeoxyribonuclease V |
#3: Protein | Mass: 67047.422 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 / Plasmid: pCDFduet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P04993, exodeoxyribonuclease V |
#4: Protein | Mass: 11661.924 Da / Num. of mol.: 2 / Mutation: L79I same as crystal structure Source method: isolated from a genetically manipulated source 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. Source: (gene. exp.) Enterobacteria phage lambda (virus) / Gene: gam, gamma / Plasmid: pET22b / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P03702 |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Source (recombinant) |
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Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||
Specimen support | Details: Grids 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 ...Details: Grids 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 Grid mesh size: 400 divisions/in. / Grid type: C-flat-1/1 | ||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K / Details: 3 ul sample applied Blot force of -4 for 1 s |
-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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 105000 X / Calibrated magnification: 37313 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Calibrated defocus min: 600 nm / Calibrated defocus max: 2900 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 0.4 sec. / Electron dose: 1.4 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 334 |
Image scans | Sampling size: 5 µm / Movie frames/image: 25 / Used frames/image: 1-25 |
-Processing
Software | Name: PHENIX / Version: 1.10.1_2155: / Classification: refinement | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: Frames were aligned using motioncorr prior to processing. | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
CTF correction | Details: CTF correction done at start of processing / Type: PHASE FLIPPING ONLY | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 134124 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122796 / Symmetry type: POINT | |||||||||||||||||||||||||||||||||||||||||||||||||||||||
Atomic model building |
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Atomic model building |
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Refine LS restraints |
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