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- PDB-8s70: Cryo-EM structure of Pseudomonas aeruginosa recombinase A (RecA) ... -

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Basic information

Entry
Database: PDB / ID: 8s70
TitleCryo-EM structure of Pseudomonas aeruginosa recombinase A (RecA) in complex with ssDNA 72mer and ATPgS
Components
  • DNA
  • Protein RecA
KeywordsDNA BINDING PROTEIN / recombinase / co-protease activator
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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / Protein RecA
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / helical reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsDe Felice, S. / Vascon, F. / Huber, S.T. / Grinzato, A. / 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
DepositionFeb 28, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 15, 2025Provider: repository / Type: Initial release
Revision 1.1Feb 12, 2025Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_last ..._citation.journal_volume / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _em_admin.last_update
Revision 1.2Mar 12, 2025Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Protein RecA
T: DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)36,7814
Polymers36,2342
Non-polymers5482
Water00
1
D: Protein RecA
T: DNA
hetero molecules
x 16


Theoretical massNumber of molelcules
Total (without water)588,50364
Polymers579,74232
Non-polymers8,76132
Water0
TypeNameSymmetry operationNumber
point symmetry operation15
identity operation1_555x,y,z1

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Components

#1: Protein Protein RecA / Recombinase A


Mass: 35062.066 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pseudomonas aeruginosa (bacteria) / Gene: recA, PA3617 / Production host: Escherichia coli (E. coli) / References: UniProt: P08280
#2: DNA chain DNA


Mass: 1171.814 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#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.: 1 / 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: helical reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1recombinase A (RecA) assembled onto ssDNA 72-mer in presence of ATPgS and Mg2+COMPLEX#1-#20MULTIPLE SOURCES
2Recombinase A (RecA)COMPLEX#11RECOMBINANT
3ssDNACOMPLEX#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Pseudomonas aeruginosa PAO1 (bacteria)208964
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
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMtris(hydroxymethyl)aminomethaneTris1
2150 mMsodium chlorideNaCl1
SpecimenConc.: 2.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298.15 K

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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: 49 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: cryoSPARC / Version: 4.2.1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Helical symmertyAngular rotation/subunit: 59.2 ° / Axial rise/subunit: 15.4 Å / Axial symmetry: C1
Particle selectionNum. of particles selected: 427064
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 202842 / Symmetry type: HELICAL
Atomic model building
IDSpaceProtocol
1REAL
2OTHER
Atomic model building
ID 3D fitting-IDSource nameType
11SwissModelin silico model
22
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00430910
ELECTRON MICROSCOPYf_angle_d0.60341892
ELECTRON MICROSCOPYf_dihedral_angle_d12.6714628
ELECTRON MICROSCOPYf_chiral_restr0.0444838
ELECTRON MICROSCOPYf_plane_restr0.0045323

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