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- PDB-8b1t: RecBCD-DNA in complex with the phage protein Abc2 -

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Basic information

Entry
Database: PDB / ID: 8b1t
TitleRecBCD-DNA in complex with the phage protein Abc2
Components
  • (RecBCD enzyme subunit ...) x 3
  • Anti-RecBCD protein 2
  • DNA (70-MER)
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 / clearance of foreign intracellular DNA / DNA 5'-3' helicase / single-stranded DNA helicase activity / recombinational repair / DNA 3'-5' helicase / 3'-5' DNA helicase activity / DNA helicase activity ...exodeoxyribonuclease V / exodeoxyribonuclease V activity / exodeoxyribonuclease V complex / clearance of foreign intracellular DNA / DNA 5'-3' helicase / single-stranded DNA helicase activity / recombinational repair / DNA 3'-5' helicase / 3'-5' DNA helicase activity / DNA helicase activity / helicase activity / response to radiation / double-strand break repair via homologous recombination / 5'-3' DNA helicase activity / DNA recombination / DNA damage response / magnesium ion binding / ATP hydrolysis activity / DNA binding / ATP binding / cytosol
Similarity search - Function
Anti-RecBCD protein 2, bacteriophage P22 / Bacteriophage P22, anti-RecBCD protein 2 / 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 ...Anti-RecBCD protein 2, bacteriophage P22 / Bacteriophage P22, anti-RecBCD protein 2 / 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 / AAA domain / DExx box DNA helicase domain superfamily / UvrD-like helicase C-terminal 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/REP helicase N-terminal domain / PD-(D/E)XK endonuclease-like domain superfamily / UvrD-like DNA helicase ATP-binding domain profile. / DNA helicase, UvrD/REP type / UvrD-like helicase, ATP-binding domain / 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
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / RecBCD enzyme subunit RecB / RecBCD enzyme subunit RecD / RecBCD enzyme subunit RecC / Anti-RecBCD protein 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Salmonella phage P22 (virus)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 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
Revision 1.1Jul 24, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: RecBCD enzyme subunit RecB
C: RecBCD enzyme subunit RecC
D: RecBCD enzyme subunit RecD
X: DNA (70-MER)
A: Anti-RecBCD protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)363,6437
Polymers363,1125
Non-polymers5312
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

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RecBCD enzyme subunit ... , 3 types, 3 molecules BCD

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


Mass: 134066.625 Da / Num. of mol.: 1 / Mutation: D1080A
Source method: isolated from a genetically manipulated source
Details: Nuclease-dead mutant D1080A / 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: pRSFduet / 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

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DNA chain / Protein , 2 types, 2 molecules XA

#4: DNA chain DNA (70-MER)


Mass: 21440.707 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: Synthesised hairpin substrate with unpaired ssDNA tails
Source: (synth.) synthetic construct (others)
#5: Protein Anti-RecBCD protein 2 / Protein abc2


Mass: 11640.210 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Salmonella phage P22 (virus) / Gene: abc2 / Plasmid: pET22b / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: P11191

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Non-polymers , 2 types, 2 molecules

#6: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


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

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Details

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
1RecBCD-Abc2-DNA complexCOMPLEXAll proteins co-expressed and purified as one complex. DNA added prior to making grids in a 1.5x molar excess#1-#50MULTIPLE SOURCES
2RecBCD enzyme subunit RecB, RecC and RecDCOMPLEX#1-#31RECOMBINANT
3DNA (70-MER)COMPLEX#41RECOMBINANT
4Anti-RecBCD protein 2COMPLEX#51RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli (E. coli)562
33synthetic construct (others)32630
44Salmonella phage P22 (virus)2908168
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21 (bacteria)511693
33synthetic construct (others)32630
44Escherichia coli BL21 (bacteria)511693
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HCl1
2100 mMSodium chlorideNaCl1
30.5 mMTCEP1
45 mMMagnesium chlorideMgCl21
52 mMADPNP1
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: RecBCD-Abc2 mixed with 1.5x molar excess of synthesised DNA substrate, 2 mM ADPNP and 5 mM MgCl2 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: 400 divisions/in. / Grid type: C-flat-2/1
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: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 56 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2500
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatchparticle selectionTemplate-based picking using reprojections
2EPUimage acquisition
4GctfCTF correction
7Coot0.9model fitting
9RELION3initial 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: 467613
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 119163 / Symmetry type: POINT
Atomic model buildingB value: 72 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model building

3D fitting-ID: 1 / Source name: PDB / Type: experimental model

IDPDB-IDPdb chain-IDAccession codeInitial refinement model-IDPdb chain residue range
15LD2

5ld2

5LD21
28B1R

8b1r

B8B1R2150-300

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