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- PDB-8ffi: Structure of tetramerized MapSPARTA upon guide RNA-mediated targe... -

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Basic information

Entry
Database: PDB / ID: 8ffi
TitleStructure of tetramerized MapSPARTA upon guide RNA-mediated target DNA binding
Components
  • TIR-APAZ
  • guide RNA
  • short pAgo
  • target DNA
KeywordsIMMUNE SYSTEM / pAgo / SPARTA / Argonuare / Oligomerization / TIR domain / NAD
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
DNA / DNA (> 10) / RNA / RNA (> 10) / Piwi domain-containing protein / TIR domain-containing protein
Similarity search - Component
Biological speciesMaribacter polysiphoniae (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsShen, Z.F. / Yang, X.Y. / Fu, T.M.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nature / Year: 2023
Title: Oligomerization-mediated activation of a short prokaryotic Argonaute.
Authors: Zhangfei Shen / Xiao-Yuan Yang / Shiyu Xia / Wei Huang / Derek J Taylor / Kotaro Nakanishi / Tian-Min Fu /
Abstract: Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P) to induce bacterial cell death. Here we present ...Although eukaryotic and long prokaryotic Argonaute proteins (pAgos) cleave nucleic acids, some short pAgos lack nuclease activity and hydrolyse NAD(P) to induce bacterial cell death. Here we present a hierarchical activation pathway for SPARTA, a short pAgo consisting of an Argonaute (Ago) protein and TIR-APAZ, an associated protein. SPARTA progresses through distinct oligomeric forms, including a monomeric apo state, a monomeric RNA-DNA-bound state, two dimeric RNA-DNA-bound states and a tetrameric RNA-DNA-bound active state. These snapshots together identify oligomerization as a mechanistic principle of SPARTA activation. The RNA-DNA-binding channel of apo inactive SPARTA is occupied by an auto-inhibitory motif in TIR-APAZ. After the binding of RNA-DNA, SPARTA transitions from a monomer to a symmetric dimer and then an asymmetric dimer, in which two TIR domains interact through charge and shape complementarity. Next, two dimers assemble into a tetramer with a central TIR cluster responsible for hydrolysing NAD(P). In addition, we observe unique features of interactions between SPARTA and RNA-DNA, including competition between the DNA 3' end and the auto-inhibitory motif, interactions between the RNA G2 nucleotide and Ago, and splaying of the RNA-DNA duplex by two loops exclusive to short pAgos. Together, our findings provide a mechanistic basis for the activation of short pAgos, a large section of the Ago superfamily.
History
DepositionDec 8, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 23, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 20, 2023Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _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-APAZ
B: short pAgo
C: guide RNA
D: target DNA
E: TIR-APAZ
F: short pAgo
G: guide RNA
H: target DNA
I: TIR-APAZ
J: guide RNA
K: target DNA
L: short pAgo
M: TIR-APAZ
N: short pAgo
O: guide RNA
P: target DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)502,85320
Polymers502,75616
Non-polymers974
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
TIR-APAZ


Mass: 53270.594 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Maribacter polysiphoniae (bacteria) / Gene: LX92_01810
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A316E683
#2: Protein
short pAgo


Mass: 58091.410 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Maribacter polysiphoniae (bacteria) / Gene: LX92_01809
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A316E3U6
#3: RNA chain
guide RNA /


Mass: 6651.949 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain
target DNA


Mass: 7675.000 Da / Num. of mol.: 4 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Structure of tetramerized MapSPARTA upon guide RNA-mediated target DNA binding
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Maribacter polysiphoniae (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
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 FIELDBright-field microscopy / Nominal defocus max: 2100 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 490999 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00333676
ELECTRON MICROSCOPYf_angle_d0.5146256
ELECTRON MICROSCOPYf_dihedral_angle_d16.3765628
ELECTRON MICROSCOPYf_chiral_restr0.0415076
ELECTRON MICROSCOPYf_plane_restr0.0045264

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