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- PDB-8v49: CryoEM structure of AriA (E393Q) sensory subunit -

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Basic information

Entry
Database: PDB / ID: 8v49
TitleCryoEM structure of AriA (E393Q) sensory subunit
ComponentsAriA antitoxin
KeywordsIMMUNE SYSTEM / Bacterial Defense system / Toxin-antitoxin system / AriAB / PARIS
Function / homologyADENOSINE-5'-TRIPHOSPHATE / :
Function and homology information
Biological speciesEscherichia coli B185 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsDeep, A. / Corbett, K.D.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM144121 United States
CitationJournal: Nature / Year: 2024
Title: Architecture and activation mechanism of the bacterial PARIS defence system.
Authors: Amar Deep / Qishan Liang / Eray Enustun / Joe Pogliano / Kevin D Corbett /
Abstract: Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race. While the mechanisms of many bacterial antiphage defence systems are known, how these systems ...Bacteria and their viruses (bacteriophages or phages) are engaged in an intense evolutionary arms race. While the mechanisms of many bacterial antiphage defence systems are known, how these systems avoid toxicity outside infection yet activate quickly after infection is less well understood. Here we show that the bacterial phage anti-restriction-induced system (PARIS) operates as a toxin-antitoxin system, in which the antitoxin AriA sequesters and inactivates the toxin AriB until triggered by the T7 phage counterdefence protein Ocr. Using cryo-electron microscopy, we show that AriA is related to SMC-family ATPases but assembles into a distinctive homohexameric complex through two oligomerization interfaces. In uninfected cells, the AriA hexamer binds to up to three monomers of AriB, maintaining them in an inactive state. After Ocr binding, the AriA hexamer undergoes a structural rearrangement, releasing AriB and allowing it to dimerize and activate. AriB is a toprim/OLD-family nuclease, the activation of which arrests cell growth and inhibits phage propagation by globally inhibiting protein translation through specific cleavage of a lysine tRNA. Collectively, our findings reveal the intricate molecular mechanisms of a bacterial defence system triggered by a phage counterdefence protein, and highlight how an SMC-family ATPase has been adapted as a bacterial infection sensor.
History
DepositionNov 28, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 26, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Aug 21, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.4Oct 16, 2024Group: Data collection / Database references / Structure summary
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: AriA antitoxin
B: AriA antitoxin
D: AriA antitoxin
C: AriA antitoxin
hetero molecules


Theoretical massNumber of molelcules
Total (without water)213,6565
Polymers213,1484
Non-polymers5071
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
AriA antitoxin


Mass: 53287.113 Da / Num. of mol.: 4 / Mutation: E393Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli B185 (bacteria) / Gene: ECDG_03487 / Production host: Escherichia coli (E. coli) / Strain (production host): Rosetta2 pLysS / References: UniProt: D6IC77
#2: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP, energy-carrying molecule*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: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: AriA hexamer / Type: COMPLEX / Details: AriA / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.30 MDa / Experimental value: YES
Source (natural)Organism: Escherichia coli B185 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: Rosetta2 pLysS
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
1250 mMSodium chlorideNaCl1
220 mMtris(hydroxymethyl)aminomethaneTris1
32 mM2-MercaptoethanolBME1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 278 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: 500 nm / Cs: 2.7 mm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5174
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Version: 1.21rc1_5127 / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 106506 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 189.9 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004713066
ELECTRON MICROSCOPYf_angle_d0.857917583
ELECTRON MICROSCOPYf_chiral_restr0.05461933
ELECTRON MICROSCOPYf_plane_restr0.00592244
ELECTRON MICROSCOPYf_dihedral_angle_d12.45064869

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