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- EMDB-19761: Cryo-EM structure of Pseudomonas aeruginosa recombinase A (RecA) ... -

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Basic information

Entry
Database: EMDB / ID: EMD-19761
TitleCryo-EM structure of Pseudomonas aeruginosa recombinase A (RecA) in complex with ssDNA 72mer and ATPgS
Map data
Sample
  • Complex: recombinase A (RecA) assembled onto ssDNA 72-mer in presence of ATPgS and Mg2+
    • Complex: Recombinase A (RecA)
      • Protein or peptide: Protein RecA
    • Complex: ssDNA
      • DNA: DNA
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
Keywordsrecombinase / co-protease activator / DNA BINDING PROTEIN
Function / homology
Function and homology information


SOS response / ATP-dependent DNA damage sensor activity / single-stranded DNA binding / DNA recombination / damaged DNA binding / DNA repair / ATP hydrolysis activity / ATP binding / cytoplasm
Similarity search - Function
DNA recombination/repair protein RecA, conserved site / DNA recombination and repair protein RecA, C-terminal / : / RecA C-terminal domain / 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/repair protein RecA, conserved site / DNA recombination and repair protein RecA, C-terminal / : / RecA C-terminal domain / 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 speciesPseudomonas aeruginosa PAO1 (bacteria) / synthetic construct (others) / Pseudomonas aeruginosa (bacteria)
Methodhelical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsDe Felice S / Vascon F / Huber ST / Grinzato A / Jakobi AJ / Cendron L
Funding support Italy, 1 items
OrganizationGrant numberCountry
Italian Ministry of Education Italy
CitationJournal: iScience / Year: 2025
Title: Snapshots of SOS response reveal structural requisites for LexA autoproteolysis.
Authors: Filippo Vascon / Sofia De Felice / Matteo Gasparotto / Stefan T Huber / Claudio Catalano / Monica Chinellato / Riccardo Mezzetti / Alessandro Grinzato / Francesco Filippini / Lorenzo Maso / ...Authors: Filippo Vascon / Sofia De Felice / Matteo Gasparotto / Stefan T Huber / Claudio Catalano / Monica Chinellato / Riccardo Mezzetti / Alessandro Grinzato / Francesco Filippini / Lorenzo Maso / Arjen J Jakobi / Laura Cendron /
Abstract: Antimicrobial resistance poses a severe threat to human health and stands out among the pathogens responsible for this emergency. The SOS response to DNA damage is crucial in bacterial evolution, ...Antimicrobial resistance poses a severe threat to human health and stands out among the pathogens responsible for this emergency. The SOS response to DNA damage is crucial in bacterial evolution, influencing resistance development and adaptability in challenging environments, especially under antibiotic exposure. Recombinase A (RecA) and the transcriptional repressor LexA are the key players that orchestrate this process, determining either the silencing or the active transcription of the genes under their control. By integrating state-of-the-art structural approaches with binding and functional assays, we elucidated the molecular events activating the SOS response in , focusing on the RecA-LexA interaction. Our findings identify the conserved determinants and strength of the interactions that allow RecA to trigger LexA autocleavage and inactivation. These results provide the groundwork for designing novel antimicrobial strategies and exploring the potential translation of -derived approaches, to address the implications of infections.
History
DepositionFeb 28, 2024-
Header (metadata) releaseJan 15, 2025-
Map releaseJan 15, 2025-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_19761.png

Downloads & links

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Map

FileDownload / File: emd_19761.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 317.568 Å		0.83 Å/pix.
x 384 pix.
= 317.568 Å		0.83 Å/pix.
x 384 pix.
= 317.568 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.827 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.105
Minimum - Maximum-0.023681452 - 0.32972655
Average (Standard dev.)0.0038203949 (±0.019199077)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 317.568 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_19761_msk_1.map
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Additional map: #2

Fileemd_19761_additional_1.map
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Additional map: #1

Fileemd_19761_additional_2.map
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Half map: #2

Fileemd_19761_half_map_1.map
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Half map: #1

Fileemd_19761_half_map_2.map
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Sample components

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Entire : recombinase A (RecA) assembled onto ssDNA 72-mer in presence of A...

EntireName: recombinase A (RecA) assembled onto ssDNA 72-mer in presence of ATPgS and Mg2+
Components
  • Complex: recombinase A (RecA) assembled onto ssDNA 72-mer in presence of ATPgS and Mg2+
    • Complex: Recombinase A (RecA)
      • Protein or peptide: Protein RecA
    • Complex: ssDNA
      • DNA: DNA
  • Ligand: MAGNESIUM ION
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

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Supramolecule #1: recombinase A (RecA) assembled onto ssDNA 72-mer in presence of A...

SupramoleculeName: recombinase A (RecA) assembled onto ssDNA 72-mer in presence of ATPgS and Mg2+
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2

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Supramolecule #2: Recombinase A (RecA)

SupramoleculeName: Recombinase A (RecA) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)

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Supramolecule #3: ssDNA

SupramoleculeName: ssDNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: synthetic construct (others)

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Macromolecule #1: Protein RecA

MacromoleculeName: Protein RecA / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria)
Molecular weightTheoretical: 35.062066 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: GDENKKRALA AALGQIERQF GKGAVMRMGD HERQAIPAIS TGSLGLDIAL GIGGLPKGRI VEIYGPESSG KTTLTLSVIA EAQKQGATC AFVDAEHALD PDYAGKLGVN VDDLLVSQPD TGEQALEITD MLVRSNAVDV IIVDSVAALV PKAEIEGEMG D AHVGLQAR ...String:
GDENKKRALA AALGQIERQF GKGAVMRMGD HERQAIPAIS TGSLGLDIAL GIGGLPKGRI VEIYGPESSG KTTLTLSVIA EAQKQGATC AFVDAEHALD PDYAGKLGVN VDDLLVSQPD TGEQALEITD MLVRSNAVDV IIVDSVAALV PKAEIEGEMG D AHVGLQAR LMSQALRKIT GNIKNANCLV IFINQIRMKI GVMFGNPETT TGGNALKFYA SVRLDIRRTG AVKEGDEVVG SE TRVKVVK NKVSPPFRQA EFQILYGKGI YRTGEIIDLG VQLGLVEKSG AWYSYQGSKI GQGKANAAKY LEDNPEIGSV LEK TIRDQL LA

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: synthetic construct (others)
Molecular weightTheoretical: 1.171814 KDa
SequenceString:
(DT)(DT)(DT)(DT)

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #4: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 1 / 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 statehelical array

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Sample preparation

Concentration2.3 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMTristris(hydroxymethyl)aminomethane
150.0 mMNaClsodium chloride
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 49.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Helical parameters - Δz: 15.4 Å
Applied symmetry - Helical parameters - Δ&Phi: 59.2 °
Applied symmetry - Helical parameters - Axial symmetry: C1 (asymmetric)
Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 202842
Segment selectionNumber selected: 427064
Startup modelType of model: INSILICO MODEL
Details: a homology model for the atomic structure of RecA-ssDNA-ATPgS complex was generated with Swissmodel
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: SwissModel / Chain - Initial model type: in silico model
RefinementSpace: REAL
Output model

PDB-8s70:
Cryo-EM structure of Pseudomonas aeruginosa recombinase A (RecA) in complex with ssDNA 72mer and ATPgS

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Atomic model buiding 2

RefinementProtocol: OTHER
Output model

PDB-8s70:
Cryo-EM structure of Pseudomonas aeruginosa recombinase A (RecA) in complex with ssDNA 72mer and ATPgS

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