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- EMDB-15231: Complex of RecF and DNA from Thermus thermophilus. -

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Basic information

Entry
Database: EMDB / ID: EMD-15231
TitleComplex of RecF and DNA from Thermus thermophilus.
Map dataSharpened map
Sample
  • Complex: Complex of RecF and DNA from Thermus thermophilus.
    • Protein or peptide: DNA replication and repair protein RecF
    • DNA: Oligo1
    • DNA: Oligo2
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


SOS response / single-stranded DNA binding / DNA replication / DNA repair / ATP binding / cytoplasm
Similarity search - Function
DNA-binding, RecF / DNA-binding RecF, domain 2 / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA replication and repair protein RecF
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria) / synthetic construct (others)
Methodsingle 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, 1 items
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, 2022-
Header (metadata) releaseApr 26, 2023-
Map releaseApr 26, 2023-
UpdateMay 31, 2023-
Current statusMay 31, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15231.png

Downloads & links

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Map

FileDownload / File: emd_15231.map.gz / Format: CCP4 / Size: 132.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.75
Minimum - Maximum-2.6875875 - 4.4743423
Average (Standard dev.)-0.00029177533 (±0.0770112)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions326326326
Spacing326326326
CellA=B=C: 280.36002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15231_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw map

Fileemd_15231_additional_1.map
AnnotationRaw map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15231_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15231_half_map_2.map
Projections & Slices
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Sample components

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Entire : Complex of RecF and DNA from Thermus thermophilus.

EntireName: Complex of RecF and DNA from Thermus thermophilus.
Components
  • Complex: Complex of RecF and DNA from Thermus thermophilus.
    • Protein or peptide: DNA replication and repair protein RecF
    • DNA: Oligo1
    • DNA: Oligo2
  • Ligand: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Complex of RecF and DNA from Thermus thermophilus.

SupramoleculeName: Complex of RecF and DNA from Thermus thermophilus. / type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)
Molecular weightTheoretical: 95.6 KDa

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Macromolecule #1: DNA replication and repair protein RecF

MacromoleculeName: DNA replication and repair protein RecF / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus HB8 (bacteria) / Strain: ATCC 27634 / DSM 579 / HB8
Molecular weightTheoretical: 37.933715 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: SMRLLLFRQR NFRNLALEAY RPPPGLSALV GANAQGKTSL LLGIHLALGG EVPLGLADLV RFGEEEAWLH AEVETELGAY RLEHRLGPG GREVLLNGKR VSLRTLWELP GSVLVSPLDL EAVLGPKEER RAYLDRLIAR FSRRYAALLS AYEKALRQRN A LLKAGGEG ...String:
SMRLLLFRQR NFRNLALEAY RPPPGLSALV GANAQGKTSL LLGIHLALGG EVPLGLADLV RFGEEEAWLH AEVETELGAY RLEHRLGPG GREVLLNGKR VSLRTLWELP GSVLVSPLDL EAVLGPKEER RAYLDRLIAR FSRRYAALLS AYEKALRQRN A LLKAGGEG LSAWDRELAR YGDEIVALRR RFLRRFAPIL REVHAALAAK EAGLRLEETA GEGVLRALEA SRAEERERGQ TL VGPHRDD LVFLLEGRPA HRFASRGEAK TLALALRLAE HRLLGEHHGE PPLLLVDEWG EELDEARRRA VLAYAQALPQ AIL AGLEAP PGVPVCSVVR GVVLCPGA

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Macromolecule #2: Oligo1

MacromoleculeName: Oligo1 / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.661912 KDa
SequenceString:
(DG)(DG)(DC)(DC)(DA)(DG)(DA)(DT)(DC)(DT) (DG)(DC)(DC)(DG)(DC)(DG)(DG)(DA)(DT)(DC) (DC)(DG)(DC)(DG)(DC)

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Macromolecule #3: Oligo2

MacromoleculeName: Oligo2 / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.395925 KDa
SequenceString:
(DG)(DC)(DG)(DC)(DG)(DG)(DA)(DT)(DC)(DC) (DG)(DC)(DG)(DG)(DC)(DA)(DG)(DA)(DT)(DC) (DT)(DG)(DG)(DC)(DC)(DT)(DG)(DA)(DT) (DT)(DG)(DC)(DG)(DG)(DT)(DA)(DC)(DA)(DG) (DA)

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Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 2 / Formula: ANP
Molecular weightTheoretical: 506.196 Da
Chemical component information

ChemComp-ANP:
PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / AMP-PNP, energy-carrying molecule analogue*YM

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClSodium chlorideSodium chloride
20.0 mMHEPESHEPES
10.0 mMMgCl2Magnesium chloride
GridModel: C-flat-2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsSample fixed with 0.05% glutaraldehyde and concentrated prior to vitrification; exact concentration cannot be estimated accurately.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7217 / Average electron dose: 41.71 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2381100
Startup modelType of model: OTHER / Details: Ab-initio reconstruction from cryoSPARC.
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.2) / Number images used: 122950
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

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

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