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- PDB-8ozc: cryoEM structure of SPARTA complex heterodimer apo -

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Basic information

Entry
Database: PDB / ID: 8ozc
TitlecryoEM structure of SPARTA complex heterodimer apo
Components
  • Piwi domain-containing protein
  • TIR domain-containing protein
KeywordsDNA BINDING PROTEIN / SPARTA / TIR / prokaryotic argonaute
Function / homology
Function and homology information


nucleic acid binding / signal transduction
Similarity search - Function
TIR domain / TIR domain profile. / Toll/interleukin-1 receptor homology (TIR) domain / Toll/interleukin-1 receptor homology (TIR) domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Piwi domain-containing protein / TIR domain-containing protein
Similarity search - Component
Biological speciesMaribacter polysiphoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsEkundayo, B. / Ni, D.C. / Lu, X.H. / Stahlberg, H.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
Swiss National Science FoundationCRSII5_177195 Switzerland
Swiss National Science FoundationIZLCZ0_206089 Switzerland
CitationJournal: Sci Adv / Year: 2023
Title: Activation mechanism of a short argonaute-TIR prokaryotic immune system.
Authors: Dongchun Ni / Xuhang Lu / Henning Stahlberg / Babatunde Ekundayo /
Abstract: Short prokaryotic argonaute (pAgo) and toll/interleukin-1 receptor/resistance protein (TIR)-analog of PAZ (APAZ) form a heterodimeric SPARTA complex that provides immunity to its prokaryotic host ...Short prokaryotic argonaute (pAgo) and toll/interleukin-1 receptor/resistance protein (TIR)-analog of PAZ (APAZ) form a heterodimeric SPARTA complex that provides immunity to its prokaryotic host through an abortive infection mechanism. Monomeric SPARTA senses foreign RNA/DNA duplexes to assemble an active tetramer resulting in cell death by nicotinamide adenine dinucleotide (oxidized form) (NAD) depletion via an unknown mechanism. We report nine structures of SPARTA in different functional states at a resolution range of 4.2 to 2.9 angstroms, revealing its activation mechanism. Inactive SPARTA monomers bind to RNA/DNA duplexes to form symmetric dimers mediated by the association of Ago subunits. The initiation of tetramer assembly induces flexibility of the TIR domains enabling a symmetry-breaking rotational movement of a TIR domain in the dimer units which facilitates the TIR oligomerization, resulting in the formation of the substrate binding pocket and the activation of the SPARTA complex's NADase activity. Our findings provide detailed structural and mechanistic insights into activating a short argonaute defense system.
History
DepositionMay 9, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 16, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2023Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last ..._citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: TIR domain-containing protein
B: Piwi domain-containing protein


Theoretical massNumber of molelcules
Total (without water)111,3622
Polymers111,3622
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area7050 Å2
ΔGint-32 kcal/mol
Surface area44100 Å2
MethodPISA

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Components

#1: Protein TIR domain-containing protein


Mass: 53270.594 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Maribacter polysiphoniae (bacteria) / Gene: LX92_01810 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A316E683
#2: Protein Piwi domain-containing protein


Mass: 58091.410 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Maribacter polysiphoniae (bacteria) / Gene: LX92_01809 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A316E3U6

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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: heter complex of short argonaute-TIR antiviral defence system
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.5 MDa / Experimental value: YES
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding 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: 2500 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7Cootmodel fitting
9Cootmodel refinement
10PHENIXmodel refinement
11cryoSPARCinitial Euler assignment
14cryoSPARC3D reconstruction
CTF correctionType: NONE
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85650 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0027662
ELECTRON MICROSCOPYf_angle_d0.57810351
ELECTRON MICROSCOPYf_dihedral_angle_d4.01983
ELECTRON MICROSCOPYf_chiral_restr0.0421114
ELECTRON MICROSCOPYf_plane_restr0.0041311

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