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- PDB-8k88: Structure of procaryotic Ago -

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Basic information

Entry
Database: PDB / ID: 8k88
TitleStructure of procaryotic Ago
Components
  • DNA (41-mer)
  • DNA/RNA (21-mer)
  • Piwi domain protein
  • Sir2 superfamily protein
KeywordsDNA/RNA/CELL INVASION / Ago / DNA/RNA / DNA BINDING PROTEIN / CELL INVASION / DNA-RNA-CELL INVASION complex
Function / homology
Function and homology information


nucleic acid binding
Similarity search - Function
SIR2-like domain / SIR2-like domain / DHS-like NAD/FAD-binding domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / DNA/RNA hybrid / DNA/RNA hybrid (> 10) / Piwi domain protein / Sir2 superfamily protein
Similarity search - Component
Biological speciesGeobacter sulfurreducens (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsGao, X. / Sun, D. / Cui, S. / Wang, Y.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Rep / Year: 2024
Title: Nucleic acid-induced NADase activation of a short Sir2-associated prokaryotic Argonaute system.
Authors: Dapeng Sun / Kaixiang Zhu / Linyue Wang / Zhixia Mu / Kang Wu / Lei Hua / Bo Qin / Xiaopan Gao / Yumei Wang / Sheng Cui /
Abstract: Inhibition of nucleic acid targets is mediated by Argonaute (Ago) proteins guided by RNA or DNA. Although the mechanisms underpinning the functions of eukaryotic and "long" prokaryotic Ago proteins ...Inhibition of nucleic acid targets is mediated by Argonaute (Ago) proteins guided by RNA or DNA. Although the mechanisms underpinning the functions of eukaryotic and "long" prokaryotic Ago proteins (pAgos) are well understood, those for short pAgos remain enigmatic. Here, we determine two cryoelectron microscopy structures of short pAgos in association with the NADase-domain-containing protein Sir2-APAZ from Geobacter sulfurreducens (GsSir2/Ago): the guide RNA-target DNA-loaded GsSir2/Ago quaternary complex (2.58 Å) and the dimer of the quaternary complex (2.93Å). These structures show that the nucleic acid binding causes profound conformational changes that result in disorder or partial dissociation of the Sir2 domain, suggesting that it adopts a NADase-active conformation. Subsequently, two RNA-/DNA-loaded GsSir2/Ago complexes form a dimer through their MID domains, further enhancing NADase activity through synergistic effects. The findings provide a structural basis for short-pAgo-mediated defense against invading nucleic acids.
History
DepositionJul 29, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 3, 2024Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: DNA (41-mer)
D: DNA/RNA (21-mer)
A: Piwi domain protein
B: Sir2 superfamily protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,1975
Polymers139,1734
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: DNA chain DNA (41-mer)


Mass: 12525.115 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: DNA/RNA hybrid DNA/RNA (21-mer)


Mass: 6675.973 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Protein Piwi domain protein


Mass: 53325.566 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Gene: GSU1361 / Production host: Expression vector pET-mod (others) / References: UniProt: Q74DF5
#4: Protein Sir2 superfamily protein


Mass: 66645.922 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Geobacter sulfurreducens (bacteria) / Gene: GSU1360 / Production host: Expression vector pET-mod (others) / References: UniProt: Q74DF6
#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / 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: Short Prokaryotic Argonaute associated Sir2 / Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightValue: 120 kDa/nm / Experimental value: YES
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Source (recombinant)Organism: Expression vector pET-mod (others)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: NICKEL/TITANIUM
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1431771 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026934
ELECTRON MICROSCOPYf_angle_d0.549594
ELECTRON MICROSCOPYf_dihedral_angle_d16.2251267
ELECTRON MICROSCOPYf_chiral_restr0.0381074
ELECTRON MICROSCOPYf_plane_restr0.0051076

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