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- PDB-8b1r: RecBCD in complex with the phage protein gp5.9 -

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Basic information

Entry
Database: PDB / ID: 8b1r
TitleRecBCD in complex with the phage protein gp5.9
Components
  • Probable RecBCD inhibitor gp5.9
  • RecBCD enzyme subunit RecB
  • RecBCD enzyme subunit RecC
  • RecBCD enzyme subunit RecD
KeywordsDNA BINDING PROTEIN / Homologous recombination / DNA repair / phage / Helicase / Nuclease / Inhibitor / Protein complex / Enzyme / DNA mimic
Function / homology
Function and homology information


exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / DNA 5'-3' helicase / clearance of foreign intracellular DNA / DNA 3'-5' helicase / recombinational repair / single-stranded DNA helicase activity / 3'-5' DNA helicase activity / DNA helicase activity ...exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / DNA 5'-3' helicase / clearance of foreign intracellular DNA / DNA 3'-5' helicase / recombinational repair / single-stranded DNA helicase activity / 3'-5' DNA helicase activity / DNA helicase activity / isomerase activity / helicase activity / double-strand break repair via homologous recombination / response to radiation / 5'-3' DNA helicase activity / DNA recombination / DNA damage response / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / cytosol
Similarity search - Function
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 / PD-(D/E)XK endonuclease-like domain, AddAB-type ...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 / PD-(D/E)XK endonuclease-like domain, AddAB-type / PD-(D/E)XK nuclease superfamily / DExx box DNA helicase domain superfamily / UvrD-like DNA helicase C-terminal domain profile. / UvrD-like DNA helicase, C-terminal / UvrD-like helicase C-terminal domain / AAA 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 / Restriction endonuclease type II-like / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / Probable RecBCD inhibitor gp5.9
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Escherichia phage T7 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWilkinson, M. / Wilkinson, O.J. / Feyerherm, C. / Fletcher, E.E. / Wigley, D.B. / Dillingham, M.S.
Funding support United Kingdom, 3items
OrganizationGrant numberCountry
Wellcome Trust100401/Z/12/Z United Kingdom
Biotechnology and Biological Sciences Research Council (BBSRC)BB/S007261/1 United Kingdom
Medical Research Council (MRC, United Kingdom) United Kingdom
CitationJournal: Elife / Year: 2022
Title: Structures of RecBCD in complex with phage-encoded inhibitor proteins reveal distinctive strategies for evasion of a bacterial immunity hub.
Authors: Martin Wilkinson / Oliver J Wilkinson / Connie Feyerherm / Emma E Fletcher / Dale B Wigley / Mark S Dillingham /
Abstract: Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, ...Following infection of bacterial cells, bacteriophage modulate double-stranded DNA break repair pathways to protect themselves from host immunity systems and prioritise their own recombinases. Here, we present biochemical and structural analysis of two phage proteins, gp5.9 and Abc2, which target the DNA break resection complex RecBCD. These exemplify two contrasting mechanisms for control of DNA break repair in which the RecBCD complex is either inhibited or co-opted for the benefit of the invading phage. Gp5.9 completely inhibits RecBCD by preventing it from binding to DNA. The RecBCD-gp5.9 structure shows that gp5.9 acts by substrate mimicry, binding predominantly to the RecB arm domain and competing sterically for the DNA binding site. Gp5.9 adopts a parallel coiled-coil architecture that is unprecedented for a natural DNA mimic protein. In contrast, binding of Abc2 does not substantially affect the biochemical activities of isolated RecBCD. The RecBCD-Abc2 structure shows that Abc2 binds to the Chi-recognition domains of the RecC subunit in a position that might enable it to mediate the loading of phage recombinases onto its single-stranded DNA products.
History
DepositionSep 12, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 28, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: RecBCD enzyme subunit RecB
C: RecBCD enzyme subunit RecC
D: RecBCD enzyme subunit RecD
P: Probable RecBCD inhibitor gp5.9
Q: Probable RecBCD inhibitor gp5.9
hetero molecules


Theoretical massNumber of molelcules
Total (without water)342,1996
Polymers342,1745
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein RecBCD enzyme subunit RecB / Exonuclease V subunit RecB / ExoV subunit RecB / Helicase/nuclease RecBCD subunit RecB


Mass: 134110.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Gene: recB, BHS81_16925, BJI68_00835, BON89_01565, BON93_20715, BvCmsHHP056_02020, C5N07_06355, C5Y87_19730, C9114_10810, DAH36_05180, DAH37_01130, E2134_07940, E5M02_13555, E5P30_22870, E5P31_10440, ...Gene: recB, BHS81_16925, BJI68_00835, BON89_01565, BON93_20715, BvCmsHHP056_02020, C5N07_06355, C5Y87_19730, C9114_10810, DAH36_05180, DAH37_01130, E2134_07940, E5M02_13555, E5P30_22870, E5P31_10440, E5P32_13260, E5P36_03520, E5P40_11525, E5P51_08340, E5S39_06465, E5S44_07560, EI021_14360, ELX85_18530, EYV17_10125, EYV18_15060, F0L67_06605, F2N31_08115, F9V24_07045, FOI11_01100, FOI11_022080, GF699_18635, GKF89_09600, GRW05_08565, HMV95_04145, HX136_04845, IH772_15520, JNP96_21695, NCTC13216_00866, NCTC9117_01550, NCTC9706_03254, RG28_07470
Plasmid: pETduet / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A024LB08, 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 / Production host: Escherichia coli BL21 (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: 66990.367 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 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P04993, exodeoxyribonuclease V
#4: Protein Probable RecBCD inhibitor gp5.9 / Gene product 5.9 / Gp5.9


Mass: 6049.601 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage T7 (virus) / Gene: 5.9 / Plasmid: pACEBAC1 / Cell line (production host): High Five / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P20406
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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
1Complex of gp5.9 bound to RecBCDCOMPLEXt7 gp5.9 purified separately and then mixed with RecBCD complex#1-#40MULTIPLE SOURCES
2RecBCD enzyme subunit RecB, RecC and RecDCOMPLEX#1-#31RECOMBINANT
3Probable RecBCD inhibitor gp5.9COMPLEX#41RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33Escherichia phage T7 (virus)10760
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21 (bacteria)511693
33Trichoplusia ni (cabbage looper)7111
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HCl1
2100 mMSodium chlorideNaCl1
30.5 mMTCEP1
45 mMMagnesium chlorideMgCl21
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: RecBCD mixed with T7 gp5.9 protein prior to making grids
Specimen supportDetails: Mixture of graphene oxide with 0.3 mM DDM detergent applied directly to grids twice before application of sample
Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Details: 1.5s blot time

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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 FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.3 sec. / Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5064

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Processing

EM software
IDNameVersionCategoryDetails
2EPUimage acquisition
4GctfCTF correction
7Coot0.9model fitting
9cryoSPARC2initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13REFMACmodel refinementRefmac jelly-body refinement
14PHENIX1.17model refinementreal-space refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1458124
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 141458 / Symmetry type: POINT
Atomic model buildingB value: 89.5 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 5MBV

5mbv

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