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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
DescriptorRecBCD enzyme subunit RecB
RecBCD enzyme subunit RecC
RecBCD enzyme subunit RecD
Host-nuclease inhibitor protein gam
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 A resolution / Single particle)
AuthorsWilkinson, M. / Chaban, Y. / Wigley, D.B.
CitationElife, 2016, 5

Elife, 2016, 5 StrPapers
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

DateDeposition: Nov 8, 2016 / Release: Jan 11, 2017

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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
Polyers353,5135
Non-polymers00
Water0
#1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (A2)25710
ΔGint (kcal/M)-97
Surface area (A2)129280
MethodPISA

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Components

#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: 3.1.11.5

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: 3.1.11.5

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: 3.1.11.5

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

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentReconstruction method: SINGLE PARTICLE

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Sample preparation

Assembly of specimen
ID
1
2
3
Component
IDNameDetailsTypeAssembly id
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.COMPLEX1
2RecBCD helicase/nuclease complexCOMPLEX2
3Lambda GamSCOMPLEX3
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.
Grid mesh size: 400 /inch. / Grid type: EMS C-Flat 1/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 / Details: 3 ul sample applied Blot force of -4 for 1 s

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Electron microscopy imaging

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 X / Calibrated magnification: 37313 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
EM image scansSampling size: 5 microns

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Processing

SoftwareName: PHENIX / Version: 1.10.1_2155: / Classification: refinement
ComputingStructure refinement: PHENIX (1.10.1_2155: phenix.real_space_refine)
3D reconstructionResolution: 3.8 A / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 122796
Atomic model building
IDRef protocolTarget criteria
1RIGID BODY FITCorrelation coefficient
2RIGID BODY FITCorrelation coefficient
Atomic model building
PDB-ID 3D fitting idID
5LD211
2UUZ22
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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