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- PDB-8amf: Cryo-EM structure of the RecA postsynaptic filament from S. pneumoniae -

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Basic information

Entry
Database: PDB / ID: 8amf
TitleCryo-EM structure of the RecA postsynaptic filament from S. pneumoniae
Components
  • (DNA) x 2
  • Protein RecA
KeywordsRECOMBINATION / Helical reconstruction / Recombinase / Streptococcus pneumoniae / double-stranded DNA
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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / Protein RecA
Similarity search - Component
Biological speciesStreptococcus pneumoniae (bacteria)
Lambdavirus lambda
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsPerry, T.N. / Fronzes, R. / Polard, P. / Hertzog, M.
Funding supportEuropean Union, 1items
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, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 21, 2024Provider: repository / Type: Initial release
Revision 1.1Mar 6, 2024Group: Advisory / Database references / Derived calculations
Category: citation / citation_author ...citation / citation_author / pdbx_validate_close_contact / struct_conn / struct_conn_type
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Protein RecA
C: DNA
D: DNA
B: Protein RecA
F: Protein RecA
G: Protein RecA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)176,20810
Polymers174,1156
Non-polymers2,0934
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Protein RecA / Recombinase A


Mass: 42007.703 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae (bacteria) / Gene: recA, spr1757 / Production host: Escherichia coli (E. coli) / References: UniProt: P0A452
#2: DNA chain DNA


Mass: 3087.110 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lambdavirus lambda / Production host: Escherichia coli (E. coli)
#3: DNA chain DNA


Mass: 2996.971 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lambdavirus lambda / Production host: Escherichia coli (E. coli)
#4: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: helical reconstruction

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Sample preparation

ComponentName: RecA postsynaptic complex from S. pneumoniae / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: EMS Lacey Carbon
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2364

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
12RELION2.1classification
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 58.62 ° / Axial rise/subunit: 14.97 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 1109194
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 715954 / Symmetry type: HELICAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00710699
ELECTRON MICROSCOPYf_angle_d0.91614470
ELECTRON MICROSCOPYf_dihedral_angle_d13.3156469
ELECTRON MICROSCOPYf_chiral_restr0.0561643
ELECTRON MICROSCOPYf_plane_restr0.0061804

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