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- PDB-8wd8: Cryo-EM structure of TtdAgo-guide DNA-target DNA complex -

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Basic information

Entry
Database: PDB / ID: 8wd8
TitleCryo-EM structure of TtdAgo-guide DNA-target DNA complex
Components
  • Argonaute family protein
  • Guide DNA
  • Target DNA
KeywordsDNA BINDING PROTEIN/DNA / DNA BINDING PROTEIN-DNA COMPLEX
Function / homology
Function and homology information


nucleic acid binding
Similarity search - Function
Argonaute PAZ domain, archaea / PAZ domain / Piwi domain / Piwi domain profile. / Piwi domain / Piwi / PAZ domain superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / Argonaute family protein
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsZhuang, L.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Mol Cell / Year: 2024
Title: Molecular mechanism for target recognition, dimerization, and activation of Pyrococcus furiosus Argonaute.
Authors: Longyu Wang / Wanping Chen / Chendi Zhang / Xiaochen Xie / Fuyong Huang / Miaomiao Chen / Wuxiang Mao / Na Yu / Qiang Wei / Lixin Ma / Zhuang Li /
Abstract: The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo- ...The Argonaute nuclease from the thermophilic archaeon Pyrococcus furiosus (PfAgo) contributes to host defense and represents a promising biotechnology tool. Here, we report the structure of a PfAgo-guide DNA-target DNA ternary complex at the cleavage-compatible state. The ternary complex is predominantly dimerized, and the dimerization is solely mediated by PfAgo at PIWI-MID, PIWI-PIWI, and PAZ-N interfaces. Additionally, PfAgo accommodates a short 14-bp guide-target DNA duplex with a wedge-type N domain and specifically recognizes 5'-phosphorylated guide DNA. In contrast, the PfAgo-guide DNA binary complex is monomeric, and the engagement of target DNA with 14-bp complementarity induces sufficient dimerization and activation of PfAgo, accompanied by movement of PAZ and N domains. A closely related Argonaute from Thermococcus thioreducens adopts a similar dimerization configuration with an additional zinc finger formed at the dimerization interface. Dimerization of both Argonautes stabilizes the catalytic loops, highlighting the important role of Argonaute dimerization in the activation and target cleavage.
History
DepositionSep 14, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Database references / Category: citation / citation_author / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Feb 28, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Argonaute family protein
T: Target DNA
G: Guide DNA
B: Argonaute family protein
U: Target DNA
H: Guide DNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)195,38913
Polymers195,1786
Non-polymers2117
Water0
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 Argonaute family protein


Mass: 87488.398 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermus thermophilus (bacteria) / Gene: A3L14_06700
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0Q2M2Z1
#2: DNA chain Target DNA


Mass: 5051.302 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)
#3: DNA chain Guide DNA


Mass: 5049.273 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) Thermus thermophilus (bacteria)
#4: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / 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

Component
IDNameTypeEntity IDParent-IDSource
1Cryo-EM structure of TtdAgo-guide DNA-target DNA complexCOMPLEX#1-#30MULTIPLE SOURCES
2DNA binding proteinDNA-binding proteinCOMPLEX#11RECOMBINANT
3DNACOMPLEX#2-#31SYNTHETIC
Source (natural)Organism: Thermus thermophilus (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
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 FIELDBright-field microscopy / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 629194 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00714071
ELECTRON MICROSCOPYf_angle_d0.71119281
ELECTRON MICROSCOPYf_dihedral_angle_d15.4762281
ELECTRON MICROSCOPYf_chiral_restr0.0482182
ELECTRON MICROSCOPYf_plane_restr0.0082209

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