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- PDB-8ab0: Complex of RecO-RecR-DNA from Thermus thermophilus. -

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Basic information

Entry
Database: PDB / ID: 8ab0
TitleComplex of RecO-RecR-DNA from Thermus thermophilus.
Components
  • DNA repair protein RecO
  • Oligo1
  • Oligo2
  • Recombination protein RecRGenetic recombination
KeywordsDNA BINDING PROTEIN / DNA Repair pathway / RecFOR pathway / Thermus thermophilus
Function / homology
Function and homology information


DNA recombination / DNA repair / DNA binding / metal ion binding
Similarity search - Function
Recombination protein O, C-terminal / Recombination protein O, RecO / DNA replication/recombination mediator RecO, N-terminal / Recombination protein O C terminal / Recombination protein O N terminal / RecR, C-terminal / RecR, helix-hairpin-helix / DNA recombination protein RecR / Recombination protein RecR, conserved site / Recombination protein RecR ...Recombination protein O, C-terminal / Recombination protein O, RecO / DNA replication/recombination mediator RecO, N-terminal / Recombination protein O C terminal / Recombination protein O N terminal / RecR, C-terminal / RecR, helix-hairpin-helix / DNA recombination protein RecR / Recombination protein RecR, conserved site / Recombination protein RecR / RecR, TOPRIM domain / RecR, Cys4-zinc finger motif / RecR protein signature. / Toprim domain / ARFGAP/RecO-like zinc finger / TOPRIM / Toprim domain profile. / TOPRIM domain / Helix-hairpin-helix DNA-binding motif, class 1 / Helix-hairpin-helix DNA-binding motif class 1 / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
DNA / DNA (> 10) / Recombination protein RecR / DNA repair protein RecO
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 6.09 Å
AuthorsNirwal, S. / Czarnocki-Cieciura, M. / Chaudhary, A. / Zajko, W. / Skowronek, K. / Chamera, S. / Figiel, M. / Nowotny, M.
Funding support Poland, 1items
OrganizationGrant numberCountry
Polish National Science Centre2017/26/A/NZ1/01098 Poland
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Mechanism of RecF-RecO-RecR cooperation in bacterial homologous recombination.
Authors: Shivlee Nirwal / Mariusz Czarnocki-Cieciura / Anuradha Chaudhary / Weronika Zajko / Krzysztof Skowronek / Sebastian Chamera / Małgorzata Figiel / Marcin Nowotny /
Abstract: In bacteria, one type of homologous-recombination-based DNA-repair pathway involves RecFOR proteins that bind at the junction between single-stranded (ss) and double-stranded (ds) DNA. They ...In bacteria, one type of homologous-recombination-based DNA-repair pathway involves RecFOR proteins that bind at the junction between single-stranded (ss) and double-stranded (ds) DNA. They facilitate the replacement of SSB protein, which initially covers ssDNA, with RecA, which mediates the search for homologous sequences. However, the molecular mechanism of RecFOR cooperation remains largely unknown. We used Thermus thermophilus proteins to study this system. Here, we present a cryo-electron microscopy structure of the RecF-dsDNA complex, and another reconstruction that shows how RecF interacts with two different regions of the tetrameric RecR ring. Lower-resolution reconstructions of the RecR-RecO subcomplex and the RecFOR-DNA assembly explain how RecO is positioned to interact with ssDNA and SSB, which is proposed to lock the complex on a ssDNA-dsDNA junction. Our results integrate the biochemical data available for the RecFOR system and provide a framework for its complete understanding.
History
DepositionJul 4, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 26, 2023Provider: repository / Type: Initial release
Revision 1.1May 3, 2023Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2May 31, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
C: Recombination protein RecR
D: Recombination protein RecR
E: Recombination protein RecR
F: Recombination protein RecR
G: DNA repair protein RecO
X: Oligo1
Y: Oligo2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)130,47610
Polymers130,2807
Non-polymers1963
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, GraFix
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Recombination protein RecR / Genetic recombination


Mass: 21323.467 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Strain: ATCC 27634 / DSM 579 / HB8 / Gene: recR, TTHA1600 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 star / References: UniProt: Q5SHY0
#2: Protein DNA repair protein RecO /


Mass: 24928.121 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Strain: ATCC 27634 / DSM 579 / HB8 / Gene: TTHA0623 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 star / References: UniProt: Q5SKM0
#3: DNA chain Oligo1


Mass: 7661.912 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain Oligo2


Mass: 12395.925 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn / 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

ComponentName: Complex of RecO-RecR-DNA from Thermus thermophilus. / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: NO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaClSodium chloride1
220 mMHEPESHEPES1
310 mMMagnesium chlorideMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample fixed with 0.05% glutaraldehyde and concentrated prior to vitrification; exact concentration cannot be estimated accurately.
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: C-flat-2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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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 / Nominal defocus max: 2700 nm / Nominal defocus min: 900 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 41.71 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7217
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassification
phenix.real_space_refinedev_4694refinement
PHENIXdev_4694refinement
EM software
IDNameVersionCategory
4CTFFINDCTF correction
12cryoSPARC3.3.23D reconstruction
13PHENIX1.20.1model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2381100
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 6.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55241 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 234.71 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00163924
ELECTRON MICROSCOPYf_angle_d0.36145472
ELECTRON MICROSCOPYf_chiral_restr0.0377720
ELECTRON MICROSCOPYf_plane_restr0.0025717
ELECTRON MICROSCOPYf_dihedral_angle_d14.7528872

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