- EMDB-3460: Cryo-EM structure of Lambda Phage protein GamS bound to RecBCD. -
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Open data
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Basic information
Entry
Database: EMDB / ID: EMD-3460
Title
Cryo-EM structure of Lambda Phage protein GamS bound to RecBCD.
Map data
Sample
RecBCD 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
Journal: 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.
Entire RecBCD complex bound to inhibitory protein lambda GamS
Entire
Name: 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
Protein
Name: 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
Component #7: protein, Host-nuclease inhibitor protein gam
Protein
Name: 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
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
Vitrification
Instrument: 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
Imaging
Microscope: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 1.4 e/Å2 / Illumination mode: FLOOD BEAM
Lens
Magnification: 105000.0 X (nominal), 37313.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200.0 - 2400.0 nm
Specimen Holder
Model: FEI TITAN KRIOS AUTOGRID HOLDER
Camera
Detector: GATAN K2 SUMMIT (4k x 4k)
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Image acquisition
Image acquisition
Number of digital images: 334 / Sampling size: 5 µm
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Image processing
Processing
Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 122796 Details: Frames were aligned using motioncorr prior to processing.
3D reconstruction
Software: 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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