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- PDB-5mbv: Cryo-EM structure of Lambda Phage protein GamS bound to RecBCD. -

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Basic information

Entry
Database: PDB / ID: 5mbv
TitleCryo-EM structure of Lambda Phage protein GamS bound to RecBCD.
Components
  • Host-nuclease inhibitor protein gam
  • RecBCD enzyme subunit RecB
  • RecBCD enzyme subunit RecC
  • RecBCD enzyme subunit RecD
KeywordsDNA 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 / DNA 5'-3' helicase / DNA 3'-5' helicase / clearance of foreign intracellular DNA / DNA translocase activity / single-stranded DNA helicase activity ...symbiont-mediated evasion of DNA end degradation by host / deoxyribonuclease inhibitor activity / exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / DNA 5'-3' helicase / DNA 3'-5' helicase / 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 / DNA endonuclease activity / helicase 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
Host-nuclease inhibitor protein Gam / Host-nuclease inhibitor Gam / Host-nuclease inhibitor Gam superfamily / Host-nuclease inhibitor protein Gam / 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 ...Host-nuclease inhibitor protein Gam / Host-nuclease inhibitor Gam / Host-nuclease inhibitor Gam superfamily / Host-nuclease inhibitor protein Gam / 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
Host-nuclease inhibitor protein gam / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / RecBCD enzyme subunit RecB
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Enterobacteria phage lambda (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsWilkinson, M. / Chaban, Y. / Wigley, D.B.
CitationJournal: 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
DepositionNov 8, 2016Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 11, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2017Group: Data collection / Experimental preparation / Refinement description
Category: em_3d_fitting / em_sample_support / em_software
Item: _em_3d_fitting.target_criteria / _em_sample_support.details ..._em_3d_fitting.target_criteria / _em_sample_support.details / _em_sample_support.grid_type / _em_software.name
Revision 1.2Mar 28, 2018Group: Structure summary / Category: struct / Item: _struct.title
Revision 1.3Oct 23, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
B: RecBCD enzyme subunit RecB
C: RecBCD enzyme subunit RecC
D: RecBCD enzyme subunit RecD
A: Host-nuclease inhibitor protein gam
E: Host-nuclease inhibitor protein gam


Theoretical massNumber of molelcules
Total (without water)353,5135
Polymers353,5135
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25710 Å2
ΔGint-97 kcal/mol
Surface area129280 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: 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
#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 / Plasmid: pRSFduet-1 / Production host: Escherichia coli BL21(DE3) (bacteria) / 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: 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 Host-nuclease inhibitor protein gam


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

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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
IDNameTypeDetailsEntity IDParent-IDSource
1RecBCD complex bound to inhibitory protein lambda GamSCOMPLEXRecBCD 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.#1-#50MULTIPLE SOURCES
2RecBCD helicase/nuclease complexCOMPLEX#1-#31RECOMBINANT
3Lambda GamSCOMPLEX#4-#51RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.35 MDaNO
220.33 MDaNO
330.02 MDaNO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Enterobacteria phage lambda (virus)10710
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
22Escherichia coli (E. coli)562BL21(DE3)Multiple
33Escherichia coli (E. coli)562BL21(DE3)pET22b
Buffer solutionpH: 7.5
Buffer component
IDConc.NameBuffer-ID
120 mMTris-HClTris1
250 mMSodium chloride1
30.5 mMTCEP-HCl1
SpecimenConc.: 1.4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 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
VitrificationInstrument: 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

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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 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage 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 scansSampling size: 5 µm / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1Gautomatchparticle selectionTemplate-free using circles with diameter of 140 Angstroms
2EPUimage acquisitionFocused every 5 um
4Gctf0.5CTF correction
7Cootmodel fitting
9RELION1.4initial Euler assignment
10RELION1.4final Euler assignment
11RELION1.4classification
12RELION1.43D reconstruction
13REFMACmodel refinementJelly body refinement
14PHENIXmodel refinementReal-space refinement
Image processingDetails: Frames were aligned using motioncorr prior to processing.
CTF correctionDetails: CTF correction done at start of processing / Type: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 134124
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122796 / Symmetry type: POINT
Atomic model building
IDProtocolTarget criteria
1RIGID BODY FITCross-correlation coefficient
2RIGID BODY FITCross-correlation coefficient
Atomic model building
IDPDB-ID 3D fitting-ID
15LD2

5ld2

1
22UUZ

2uuz

2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00124635
ELECTRON MICROSCOPYf_angle_d0.42733407
ELECTRON MICROSCOPYf_dihedral_angle_d15.51414917
ELECTRON MICROSCOPYf_chiral_restr0.0363657
ELECTRON MICROSCOPYf_plane_restr0.0034402

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