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- PDB-8qlp: CryoEM structure of the RNA/DNA bound SPARTA (BabAgo/TIR-APAZ) te... -

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Basic information

Entry
Database: PDB / ID: 8qlp
TitleCryoEM structure of the RNA/DNA bound SPARTA (BabAgo/TIR-APAZ) tetrameric complex
Components
  • DNA (5'-D(*AP*CP*TP*AP*AP*TP*AP*GP*AP*TP*TP*AP*GP*AP*GP*CP*CP*GP*TP*C)-3')
  • RNA (5'-R(*AP*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
  • Short prokaryotic Argonaute
  • Toll/interleukin-1 receptor domain-containing protein
KeywordsIMMUNE SYSTEM / Prokaryotic Argonaute / TIR domain / RNA binding protein / DNA binding protein / Oligomerization / NADase activity
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesBacillales bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsFinocchio, G. / Koopal, B. / Potocnik, A. / Heijstek, C. / Jinek, M. / Swarts, D.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)European Union
CitationJournal: Nucleic Acids Res / Year: 2024
Title: Target DNA-dependent activation mechanism of the prokaryotic immune system SPARTA.
Authors: Giada Finocchio / Balwina Koopal / Ana Potocnik / Clint Heijstek / Adrie H Westphal / Martin Jinek / Daan C Swarts /
Abstract: In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains ...In both prokaryotic and eukaryotic innate immune systems, TIR domains function as NADases that degrade the key metabolite NAD+ or generate signaling molecules. Catalytic activation of TIR domains requires oligomerization, but how this is achieved varies in distinct immune systems. In the Short prokaryotic Argonaute (pAgo)/TIR-APAZ (SPARTA) immune system, TIR NADase activity is triggered upon guide RNA-mediated recognition of invading DNA by an unknown mechanism. Here, we describe cryo-EM structures of SPARTA in the inactive monomeric and target DNA-activated tetrameric states. The monomeric SPARTA structure reveals that in the absence of target DNA, a C-terminal tail of TIR-APAZ occupies the nucleic acid binding cleft formed by the pAgo and TIR-APAZ subunits, inhibiting SPARTA activation. In the active tetrameric SPARTA complex, guide RNA-mediated target DNA binding displaces the C-terminal tail and induces conformational changes in pAgo that facilitate SPARTA-SPARTA dimerization. Concurrent release and rotation of one TIR domain allow it to form a composite NADase catalytic site with the other TIR domain within the dimer, and generate a self-complementary interface that mediates cooperative tetramerization. Combined, this study provides critical insights into the structural architecture of SPARTA and the molecular mechanism underlying target DNA-dependent oligomerization and catalytic activation.
History
DepositionSep 20, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.0Jan 31, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 31, 2024Data content type: Additional map / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jan 31, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Jan 31, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.1Mar 6, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jul 9, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details
Item: _em_admin.last_update / _em_software.name / _pdbx_entry_details.has_protein_modification
Revision 1.1Jul 9, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Toll/interleukin-1 receptor domain-containing protein
B: Short prokaryotic Argonaute
C: RNA (5'-R(*AP*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
D: DNA (5'-D(*AP*CP*TP*AP*AP*TP*AP*GP*AP*TP*TP*AP*GP*AP*GP*CP*CP*GP*TP*C)-3')
E: Toll/interleukin-1 receptor domain-containing protein
F: Short prokaryotic Argonaute
G: RNA (5'-R(*AP*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
H: DNA (5'-D(*AP*CP*TP*AP*AP*TP*AP*GP*AP*TP*TP*AP*GP*AP*GP*CP*CP*GP*TP*C)-3')
I: Toll/interleukin-1 receptor domain-containing protein
J: Short prokaryotic Argonaute
K: RNA (5'-R(*AP*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
L: DNA (5'-D(*AP*CP*TP*AP*AP*TP*AP*GP*AP*TP*TP*AP*GP*AP*GP*CP*CP*GP*TP*C)-3')
M: Toll/interleukin-1 receptor domain-containing protein
N: Short prokaryotic Argonaute
O: RNA (5'-R(*AP*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
P: DNA (5'-D(*AP*CP*TP*AP*AP*TP*AP*GP*AP*TP*TP*AP*GP*AP*GP*CP*CP*GP*TP*C)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)501,25920
Polymers501,16216
Non-polymers974
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
Toll/interleukin-1 receptor domain-containing protein / TIR-APAZ


Mass: 53052.355 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillales bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#2: Protein
Short prokaryotic Argonaute / SPAR


Mass: 57911.199 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillales bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#3: RNA chain
RNA (5'-R(*AP*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')


Mass: 6651.949 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillales bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#4: DNA chain
DNA (5'-D(*AP*CP*TP*AP*AP*TP*AP*GP*AP*TP*TP*AP*GP*AP*GP*CP*CP*GP*TP*C)-3')


Mass: 7675.000 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bacillales bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
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

ComponentName: RNA/DNA bound SPARTA (BabAgo/TIR-APAZ) tetrameric complex
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Bacillales bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: 2400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 59.347 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60319 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00416418
ELECTRON MICROSCOPYf_angle_d0.54322540
ELECTRON MICROSCOPYf_dihedral_angle_d12.4912732
ELECTRON MICROSCOPYf_chiral_restr0.0392452
ELECTRON MICROSCOPYf_plane_restr0.0042578

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