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- PDB-8j8h: SPARTA monomer bound with guide-target, state 2 -

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Basic information

Entry
Database: PDB / ID: 8j8h
TitleSPARTA monomer bound with guide-target, state 2
Components
  • DNA (25-MER)
  • Piwi domain-containing protein
  • RNA (5'-R(P*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
  • TIR domain-containing protein
KeywordsDNA BINDING PROTEIN/DNA/RNA / SPARTA / Ago / Tir / DNA BINDING PROTEIN-DNA-RNA complex
Function / homology
Function and homology information


nucleic acid binding / signal transduction
Similarity search - Function
TIR domain / 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
DNA / DNA (> 10) / RNA / RNA (> 10) / Piwi domain-containing protein / TIR domain-containing protein
Similarity search - Component
Biological speciesThermoflavifilum thermophilum (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsLi, Z.X. / Guo, L.J. / Huang, P.P. / Xiao, Y.B. / Chen, M.R.
Funding support China, 5items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)Nos.2018YFA0902000 China
Ministry of Science and Technology (MoST, China)ZX-2021ZD0203404 China
National Natural Science Foundation of China (NSFC)Nos. 32271330 China
National Natural Science Foundation of China (NSFC)Nos. 32000889 China
National Natural Science Foundation of China (NSFC)31970547 China
CitationJournal: Nat Chem Biol / Year: 2024
Title: Auto-inhibition and activation of a short Argonaute-associated TIR-APAZ defense system.
Authors: Lijie Guo / Pingping Huang / Zhaoxing Li / Young-Cheul Shin / Purui Yan / Meiling Lu / Meirong Chen / Yibei Xiao /
Abstract: Short prokaryotic Ago accounts for most prokaryotic Argonaute proteins (pAgos) and is involved in defending bacteria against invading nucleic acids. Short pAgo associated with TIR-APAZ (SPARTA) has ...Short prokaryotic Ago accounts for most prokaryotic Argonaute proteins (pAgos) and is involved in defending bacteria against invading nucleic acids. Short pAgo associated with TIR-APAZ (SPARTA) has been shown to oligomerize and deplete NAD upon guide-mediated target DNA recognition. However, the molecular basis of SPARTA inhibition and activation remains unknown. In this study, we determined the cryogenic electron microscopy structures of Crenotalea thermophila SPARTA in its inhibited, transient and activated states. The SPARTA monomer is auto-inhibited by its acidic tail, which occupies the guide-target binding channel. Guide-mediated target binding expels this acidic tail and triggers substantial conformational changes to expose the Ago-Ago dimerization interface. As a result, SPARTA assembles into an active tetramer, where the four TIR domains are rearranged and packed to form NADase active sites. Together with biochemical evidence, our results provide a panoramic vision explaining SPARTA auto-inhibition and activation and expand understanding of pAgo-mediated bacterial defense systems.
History
DepositionMay 1, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Mar 6, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2024Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Piwi domain-containing protein
B: TIR domain-containing protein
E: RNA (5'-R(P*UP*GP*AP*CP*GP*GP*CP*UP*CP*UP*AP*AP*UP*CP*UP*AP*UP*UP*AP*GP*U)-3')
F: DNA (25-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)133,0195
Polymers132,9944
Non-polymers241
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Piwi domain-containing protein


Mass: 61857.793 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoflavifilum thermophilum (bacteria)
Gene: SAMN05660895_1671 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A1I7NFD7
#2: Protein TIR domain-containing protein


Mass: 56809.668 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoflavifilum thermophilum (bacteria)
Gene: SAMN05660895_1670 / Production host: Escherichia coli BL21 (bacteria) / References: UniProt: A0A1I7NFG5
#3: RNA chain RNA (5'-R(P*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.: 1 / Source method: obtained synthetically / Source: (synth.) Thermoflavifilum thermophilum (bacteria)
#4: DNA chain DNA (25-MER)


Mass: 7675.000 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermoflavifilum thermophilum (bacteria)
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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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: Short ago complexed with TIR-APAZ / Type: COMPLEX / Entity ID: #3-#4, #1-#2 / Source: MULTIPLE SOURCES
Source (natural)Organism: Thermoflavifilum thermophilum (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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 117819 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0047122
ELECTRON MICROSCOPYf_angle_d0.699787
ELECTRON MICROSCOPYf_dihedral_angle_d17.1081199
ELECTRON MICROSCOPYf_chiral_restr0.0451050
ELECTRON MICROSCOPYf_plane_restr0.0051106

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