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- EMDB-15524: Cryo-EM structure of the RecA presynaptic filament from S.pneumoniae -
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Open data
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Basic information
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Title | Cryo-EM structure of the RecA presynaptic filament from S.pneumoniae | |||||||||
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![]() | Helical reconstruction / Recombinase / Streptococcus pneumoniae / single-stranded DNA / RECOMBINATION | |||||||||
Function / homology | ![]() establishment of competence for transformation / ATP-dependent DNA damage sensor activity / SOS response / single-stranded DNA binding / DNA recombination / damaged DNA binding / DNA repair / ATP hydrolysis activity / ATP binding / cytosol Similarity search - Function | |||||||||
Biological species | ![]() ![]() ![]() | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.9 Å | |||||||||
![]() | Perry TN / Fronzes R / Polard P / Hertzog M | |||||||||
Funding support | European Union, 1 items
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![]() | ![]() Title: Assembly mechanism and cryoEM structure of RecA recombination nucleofilaments from Streptococcus pneumoniae. Authors: Maud Hertzog / Thomas Noé Perry / Pauline Dupaigne / Sandra Serres / Violette Morales / Anne-Lise Soulet / Jason C Bell / Emmanuel Margeat / Stephen C Kowalczykowski / Eric Le Cam / Rémi ...Authors: Maud Hertzog / Thomas Noé Perry / Pauline Dupaigne / Sandra Serres / Violette Morales / Anne-Lise Soulet / Jason C Bell / Emmanuel Margeat / Stephen C Kowalczykowski / Eric Le Cam / Rémi Fronzes / Patrice Polard / ![]() ![]() Abstract: RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic ...RecA-mediated homologous recombination (HR) is a key mechanism for genome maintenance and plasticity in bacteria. It proceeds through RecA assembly into a dynamic filament on ssDNA, the presynaptic filament, which mediates DNA homology search and ordered DNA strand exchange. Here, we combined structural, single molecule and biochemical approaches to characterize the ATP-dependent assembly mechanism of the presynaptic filament of RecA from Streptococcus pneumoniae (SpRecA), in comparison to the Escherichia coli RecA (EcRecA) paradigm. EcRecA polymerization on ssDNA is assisted by the Single-Stranded DNA Binding (SSB) protein, which unwinds ssDNA secondary structures that block EcRecA nucleofilament growth. We report by direct microscopic analysis of SpRecA filamentation on ssDNA that neither of the two paralogous pneumococcal SSBs could assist the extension of SpRecA nucleopolymers. Instead, we found that the conserved RadA helicase promotes SpRecA nucleofilamentation in an ATP-dependent manner. This allowed us to solve the atomic structure of such a long native SpRecA nucleopolymer by cryoEM stabilized with ATPγS. It was found to be equivalent to the crystal structure of the EcRecA filament with a marked difference in how RecA mediates nucleotide orientation in the stretched ssDNA. Then, our results show that SpRecA and EcRecA HR activities are different, in correlation with their distinct ATP-dependent ssDNA binding modes. | |||||||||
History |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 20.3 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 16.8 KB 16.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 6.5 KB | Display | ![]() |
Images | ![]() | 54.1 KB | ||
Filedesc metadata | ![]() | 6 KB | ||
Others | ![]() ![]() | 5.6 MB 5.6 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 757.8 KB | Display | ![]() |
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Full document | ![]() | 757.4 KB | Display | |
Data in XML | ![]() | 12.2 KB | Display | |
Data in CIF | ![]() | 16.9 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8amdMC ![]() 8amfC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Voxel size | X=Y=Z: 1.216 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #1
File | emd_15524_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_15524_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Presynaptic filament from S.pneumoniae
Entire | Name: Presynaptic filament from S.pneumoniae |
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Components |
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-Supramolecule #1: Presynaptic filament from S.pneumoniae
Supramolecule | Name: Presynaptic filament from S.pneumoniae / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2 / Details: RecA protein forms a dynamic filament on ssDNA |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Protein RecA
Macromolecule | Name: Protein RecA / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 42.007703 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MAKKPKKLEE ISKKFGAERE KALNDALKLI EKDFGKGSIM RLGERAEQKV QVMSSGSLAL DIALGSGGYP KGRIIEIYGP ESSGKTTVA LHAVAQAQKE GGIAAFIDAE HALDPAYAAA LGVNIDELLL SQPDSGEQGL EIAGKLIDSG AVDLVVVDSV A ALVPRAEI ...String: MAKKPKKLEE ISKKFGAERE KALNDALKLI EKDFGKGSIM RLGERAEQKV QVMSSGSLAL DIALGSGGYP KGRIIEIYGP ESSGKTTVA LHAVAQAQKE GGIAAFIDAE HALDPAYAAA LGVNIDELLL SQPDSGEQGL EIAGKLIDSG AVDLVVVDSV A ALVPRAEI DGDIGDSHVG LQARMMSQAM RKLGASINKT KTIAIFINQL REKVGVMFGN PETTPGGRAL KFYASVRLDV RG NTQIKGT GDQKETNVGK ETKIKVVKNK VAPPFKEAVV EIMYGEGISK TGELLKIASD LDIIKKAGAW YSYKDEKIGQ GSE NAKKYL AEHPEIFDEI DKQVRSKFGL IDGEEVSEQD TENKKDEPKK EEAVNEEVPL DLGDELEIEI EE UniProtKB: Protein RecA |
-Macromolecule #2: DNA
Macromolecule | Name: DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 3.605356 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT) |
-Macromolecule #3: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 3 / Number of copies: 4 / Formula: AGS |
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Molecular weight | Theoretical: 523.247 Da |
Chemical component information | ![]() ChemComp-AGS: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | helical reconstruction |
Aggregation state | filament |
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Sample preparation
Concentration | 0.2 mg/mL |
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Buffer | pH: 7.5 |
Grid | Model: EMS Lacey Carbon / Material: COPPER |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2896 / Average electron dose: 36.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 190000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |