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- PDB-8a8j: Complex of RecF and DNA from Thermus thermophilus. -

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Basic information

Entry
Database: PDB / ID: 8a8j
TitleComplex of RecF and DNA from Thermus thermophilus.
Components
  • DNA replication and repair protein RecF
  • Oligo1
  • Oligo2
KeywordsDNA BINDING PROTEIN / DNA Repair pathway / RecFOR pathway / Thermus thermophilus
Function / homology
Function and homology information


SOS response / DNA synthesis involved in DNA repair / double-strand break repair / single-stranded DNA binding / DNA replication / ATP binding / cytoplasm
Similarity search - Function
DNA-binding, RecF / DNA-binding RecF, domain 2 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA replication and repair protein RecF
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 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
DepositionJun 23, 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
Revision 1.3Jul 24, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DNA replication and repair protein RecF
B: DNA replication and repair protein RecF
X: Oligo1
Y: Oligo2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,9868
Polymers95,9254
Non-polymers1,0614
Water00
1


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

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Components

#1: Protein DNA replication and repair protein RecF


Mass: 37933.715 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus HB8 (bacteria) / Strain: ATCC 27634 / DSM 579 / HB8 / Gene: recF / Production host: Escherichia coli (E. coli) / Strain (production host): Arctic Express / References: UniProt: Q5SLM9
#2: DNA chain Oligo1


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


Mass: 12395.925 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-ANP / PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER


Mass: 506.196 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H17N6O12P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: AMP-PNP, energy-carrying molecule analogue*YM
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / 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

ComponentName: Complex of RecF and DNA from Thermus thermophilus. / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.0956 MDa / Experimental value: NO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: ArcticExpress strain
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMSodium chlorideNaCl1
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 FIELD / 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_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameVersionCategory
4CTFFINDCTF correction
12cryoSPARC3.3.23D reconstruction
13PHENIX1.19.2model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2381100
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 122950 / 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: 43.63 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00336353
ELECTRON MICROSCOPYf_angle_d0.54828773
ELECTRON MICROSCOPYf_chiral_restr0.0369977
ELECTRON MICROSCOPYf_plane_restr0.00461011
ELECTRON MICROSCOPYf_dihedral_angle_d18.91561175

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