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- EMDB-15525: Cryo-EM structure of the RecA postsynaptic filament from S. pneumoniae -

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Basic information

Entry
Database: EMDB / ID: EMD-15525
TitleCryo-EM structure of the RecA postsynaptic filament from S. pneumoniae
Map data
Sample
  • Complex: RecA postsynaptic complex from S. pneumoniae
    • Protein or peptide: Protein RecA
    • DNA: DNA
    • DNA: DNA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
KeywordsHelical reconstruction / Recombinase / Streptococcus pneumoniae / double-stranded DNA / RECOMBINATION
Function / homology
Function and homology information


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
: / : / RecA C-terminal domain / DNA recombination/repair protein RecA, conserved site / DNA recombination and repair protein RecA, C-terminal / recA signature. / DNA recombination and repair protein RecA / recA bacterial DNA recombination protein / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. ...: / : / RecA C-terminal domain / DNA recombination/repair protein RecA, conserved site / DNA recombination and repair protein RecA, C-terminal / recA signature. / DNA recombination and repair protein RecA / recA bacterial DNA recombination protein / DNA recombination and repair protein RecA, monomer-monomer interface / RecA family profile 2. / DNA recombination and repair protein RecA-like, ATP-binding domain / RecA family profile 1. / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Biological speciesStreptococcus pneumoniae (bacteria) / Lambdavirus lambda
Methodhelical reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsPerry TN / Fronzes R / Polard P / Hertzog M
Funding supportEuropean Union, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)725554European Union
CitationJournal: Nucleic Acids Res / Year: 2023
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
DepositionAug 3, 2022-
Header (metadata) releaseFeb 21, 2024-
Map releaseFeb 21, 2024-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15525.png

Downloads & links

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Map

FileDownload / File: emd_15525.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy AUTHOR: 2.5
Minimum - Maximum-9.055714 - 15.755357999999999
Average (Standard dev.)0.000000000006449 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 198.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_15525_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_15525_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : RecA postsynaptic complex from S. pneumoniae

EntireName: RecA postsynaptic complex from S. pneumoniae
Components
  • Complex: RecA postsynaptic complex from S. pneumoniae
    • Protein or peptide: Protein RecA
    • DNA: DNA
    • DNA: DNA
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: RecA postsynaptic complex from S. pneumoniae

SupramoleculeName: RecA postsynaptic complex from S. pneumoniae / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Streptococcus pneumoniae (bacteria)

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Macromolecule #1: Protein RecA

MacromoleculeName: Protein RecA / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Molecular weightTheoretical: 42.007703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #2: DNA

MacromoleculeName: DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lambdavirus lambda
Molecular weightTheoretical: 3.08711 KDa
SequenceString:
(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)

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Macromolecule #3: DNA

MacromoleculeName: DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Lambdavirus lambda
Molecular weightTheoretical: 2.996971 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)

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Macromolecule #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 4 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Experimental details

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Structure determination

Methodcryo EM
Processinghelical reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 7.5
GridModel: EMS Lacey Carbon / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2364 / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 14.97 Å
Applied symmetry - Helical parameters - Δ&Phi: 58.62 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 715954
Segment selectionNumber selected: 1109194 / Software - Name: RELION (ver. 2.1)
Startup modelType of model: OTHER
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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