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

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Database: PDB / ID: 5mbv
TitleCryo-EM structure of Lambda Phage protein GamS bound to RecBCD
DescriptorHost-nuclease inhibitor protein gam
(RecBCD enzyme subunit ...) x 3
KeywordsDNA BINDING PROTEIN / Inhibitor / Complex / DNA repair / Helicase/Nuclease / DNA binding protein
Specimen sourceEscherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
Enterobacteria phage lambda / virus
MethodElectron microscopy (3.8 Å resolution / Particle / Single particle)
AuthorsWilkinson, M. / Chaban, Y. / Wigley, D.B.
CitationElife, 2016, 5

Elife, 2016, 5 Yorodumi Papers
Structural basis for the inhibition of RecBCD by Gam and its synergistic antibacterial effect with quinolones.
Martin Wilkinson / Lucy Troman / Wan Ak Wan Nur Ismah / Yuriy Chaban / Matthew B Avison / Mark S Dillingham / Dale B Wigley

Validation Report
SummaryFull reportAbout validation report
DateDeposition: Nov 8, 2016 / Release: Jan 11, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Jan 11, 2017Structure modelrepositoryInitial release
1.1Aug 23, 2017Structure modelData collection / Experimental preparation / Refinement descriptionem_3d_fitting / em_sample_support / em_software_em_3d_fitting.target_criteria / _em_sample_support.details / _em_sample_support.grid_type / _em_software.name

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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

TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)25710
ΔGint (kcal/M)-97
Surface area (Å2)129280


#1: Polypeptide(L)RecBCD enzyme subunit RecB / 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: (gene. exp.) Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
References: UniProt: P08394, EC:

Cellular component

Molecular function

Biological process

#2: Polypeptide(L)RecBCD enzyme subunit RecC / 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: (gene. exp.) Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
References: UniProt: P07648, EC:

Cellular component

Molecular function

Biological process

#3: Polypeptide(L)RecBCD enzyme subunit RecD / 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: (gene. exp.) Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 /
References: UniProt: P04993, EC:

Cellular component

Molecular function

Biological process

#4: Polypeptide(L)Host-nuclease inhibitor protein gam

Mass: 11661.924 Da / Num. of mol.: 2
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.
Mutation: L79I same as crystal structure / Source: (gene. exp.) Enterobacteria phage lambda / virus / References: UniProt: P03702

Molecular function

Biological process

Experimental details


EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

Sample preparation



IDNameDetailsEntity IDParent IDSource
1RecBCD complex bound to inhibitory protein lambda GamSRecBCD 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, 2, 3, 4, 50MULTIPLE SOURCES
2RecBCD helicase/nuclease complex1, 2, 31RECOMBINANT
3Lambda GamS4, 51RECOMBINANT
Molecular weight
IDValueUnitsEntity assembly IDExperimental value
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
22562Escherichia coli
3310710Enterobacteria phage lambda
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganismPlasmidStrain
22562Escherichia coliMultipleBL21(DE3)
33562Escherichia colipET22bBL21(DE3)
Buffer solutionpH: 7.5
Buffer component
IDConc.UnitsNameBuffer ID
250mMSodium chloride1
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 hydrophilic. Grids were washed with 5 drops of water prior to use. EMS
Grid mesh size: 400 / Grid type: C-flat-1/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 kelvins / Details: 3 ul sample applied Blot force of -4 for 1 s

Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 / Calibrated magnification: 37313 / 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 mm / 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) / Number of grids imaged: 1 / Number of real images: 334
Image scansSampling size: 5 microns / Movie frames/image: 25 / Used frames/image: 1-25


SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
EM software
IDNameVersionCategoryDetailsImage processing IDImaging IDFitting ID
1GautomatchPARTICLE SELECTIONTemplate-free using circles with diameter of 140 Angstroms1
2EPUIMAGE ACQUISITIONFocused every 5 um1
13REFMACMODEL REFINEMENTJelly body refinement1
14PHENIXMODEL REFINEMENTReal-space refinement1
Image processingDetails: Frames were aligned using motioncorr prior to processing.
CTF correctionDetails: CTF correction done at start of processing / Type: PHASE FLIPPING ONLY
Particle selectionNumber of particles selected: 134124
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 122796 / Symmetry type: POINT
Atomic model building
IDRef protocolTarget criteria
1RIGID BODY FITCross-correlation coefficient
2RIGID BODY FITCross-correlation coefficient
Atomic model building
IDPDB-ID 3D fitting ID
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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