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- PDB-8s7g: Cryo-EM structure of Pseudomonas aeruginosa Recombinase A (RecA) ... -

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Basic information

Entry
Database: PDB / ID: 8s7g
TitleCryo-EM structure of Pseudomonas aeruginosa Recombinase A (RecA) in complex with LexAS125A mutant
Components
  • DNA (36-MER)
  • LexA repressor
  • Protein RecA
KeywordsTRANSCRIPTION / protease / antibiotic resistance / SOS response
Function / homology
Function and homology information


repressor LexA / SOS response / DNA-binding transcription repressor activity / ATP-dependent DNA damage sensor activity / protein-DNA complex / single-stranded DNA binding / DNA recombination / sequence-specific DNA binding / damaged DNA binding / DNA replication ...repressor LexA / SOS response / DNA-binding transcription repressor activity / ATP-dependent DNA damage sensor activity / protein-DNA complex / single-stranded DNA binding / DNA recombination / sequence-specific DNA binding / damaged DNA binding / DNA replication / serine-type endopeptidase activity / DNA repair / negative regulation of DNA-templated transcription / regulation of DNA-templated transcription / ATP hydrolysis activity / proteolysis / ATP binding / cytoplasm
Similarity search - Function
LexA repressor, DNA-binding domain / Transcription regulator LexA / LexA DNA binding domain / Peptidase S24, LexA-like / : / LexA-like / Peptidase S24/S26A/S26B/S26C / Peptidase S24-like / LexA/Signal peptidase-like superfamily / DNA recombination/repair protein RecA, conserved site ...LexA repressor, DNA-binding domain / Transcription regulator LexA / LexA DNA binding domain / Peptidase S24, LexA-like / : / LexA-like / Peptidase S24/S26A/S26B/S26C / Peptidase S24-like / LexA/Signal peptidase-like superfamily / 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 N-terminal domain / 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. / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily / 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 / DNA (> 10) / Protein RecA / LexA repressor
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.43 Å
AuthorsDe Felice, S. / Vascon, F. / Huber, S.T. / Catalano, C. / Jakobi, A.J. / Cendron, L.
Funding support Italy, 1items
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
DepositionMar 1, 2024Deposition site: PDBE / Processing site: PDBE
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: LexA repressor
B: LexA repressor
D: Protein RecA
E: Protein RecA
F: Protein RecA
G: Protein RecA
H: Protein RecA
I: Protein RecA
J: Protein RecA
K: Protein RecA
L: Protein RecA
T: DNA (36-MER)
C: Protein RecA
M: Protein RecA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)491,04236
Polymers485,01914
Non-polymers6,02322
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein LexA repressor


Mass: 23407.885 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: lexA, PA3007 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P37452, repressor LexA
#2: Protein
Protein RecA / Recombinase A


Mass: 38845.184 Da / Num. of mol.: 11
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: recA, PA3617 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: P08280
#3: DNA chain DNA (36-MER)


Mass: 10905.983 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 11 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: 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.: 11 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: HELICAL ARRAY / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of Pseudomonas aeruginosa Recombinase A (RecA) in complex with LexAS125A mutantCOMPLEX#1-#30MULTIPLE SOURCES
2Recombinase A (RecA) and LexA repressorCOMPLEX#1-#21RECOMBINANT
3ssDNACOMPLEX#31RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pseudomonas aeruginosa (bacteria)287
33synthetic construct (others)32630
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21(DE3) (bacteria)469008
33synthetic construct (others)32630
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
12cryoSPARC4.2.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 561719
3D reconstructionResolution: 3.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 164165 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00530463
ELECTRON MICROSCOPYf_angle_d0.52741311
ELECTRON MICROSCOPYf_dihedral_angle_d11.554589
ELECTRON MICROSCOPYf_chiral_restr0.0424771
ELECTRON MICROSCOPYf_plane_restr0.0055235

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