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- PDB-9v7z: Structure of the Argonaute protein from Verrucomicrobia bacterium... -

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Basic information

Entry
Database: PDB / ID: 9v7z
TitleStructure of the Argonaute protein from Verrucomicrobia bacterium in complex with guide DNA and target RNA in target-released state
Components
  • DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T)-3')
  • RNA (5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*A)-3')
  • VbAgo
KeywordsRNA BINDING PROTEIN/DNA/RNA / mesophilic prokaryotic Argonaute / RNA BINDING PROTEIN-DNA-RNA complex
Function / homologyDNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesVerrucomicrobiota bacterium (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsWu, S.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)240801001003 China
CitationJournal: Nat Commun / Year: 2026
Title: Structural insights into C-terminus-mediated RNA target cleavage by a mesophilic prokaryotic argonaute.
Authors: Taiyu Chen / Xin Tao / Shunshun Li / Qingmiao Duanmu / Jiening Wang / Kai Chen / Kangle Mu / Hong Yang / Yu Li / Yang Liu / Lixin Ma / Shan Wu /
Abstract: Prokaryotic Argonaute proteins (pAgos) are programmable nucleases that always utilize DNA guides to cleave DNA targets. Recent studies show that some pAgos preferentially utilize DNA guides to cleave ...Prokaryotic Argonaute proteins (pAgos) are programmable nucleases that always utilize DNA guides to cleave DNA targets. Recent studies show that some pAgos preferentially utilize DNA guides to cleave RNA targets rather than DNA targets. VbAgo, derived from a Verrucomicrobia bacterium, is a nuclease capable of specifically cleaving single-stranded RNA and highly structured RNA substrates at 37 °C, making it an ideal candidate for developing RNA manipulation toolkits. An in-depth investigation of its mechanism contributes to understanding the functional characteristics of gDtR-type Ago proteins. Here, we present cryo-electron microscopy structures of VbAgo, the VbAgo-guide DNA binary complex, multiple wild-type VbAgo-guide DNA-target RNA ternary complexes, and the catalytically inactive mutant (VbAgo-DM) guide DNA-target RNA ternary complex, with resolutions ranging from 2.5 to 3.2 Å. By integrating these cryo-EM structures with biochemical data, we elucidate the entire catalytic process of VbAgo, revealing its unique C-terminal regulatory mechanism. Specifically, in its apo state, VbAgo's C-terminus occupies the nucleic acid binding channel, partially impeding its catalytic activity while enhancing its stability. The binding of guide DNA displaces the C-terminus, and subsequent binding of target RNA, along with conformational changes in the N-terminal and PAZ domains, facilitates VbAgo dimerization. Following this, the C-terminus transitions from a loop to a helix, enabling maturation of the catalytic center and inducing movements in the MID-PIWI' interactions at the dimer interfaces, ultimately leading to dimer dissociation. Concurrently, cleavage of the target RNA and subsequent product release occur, after which VbAgo reverts to its binary state to initiate the next cleavage cycle. Moreover, we demonstrate that VbAgo exhibits guide DNA mediated RNA knockdown activity in mammalian cells. In summary, our study provides a comprehensive understanding of the molecular mechanisms governing self-inhibition, guide binding, target recognition, and product release in VbAgo. These findings offer valuable insights into the diverse mechanisms of pAgos, broadening their functional scope and enhancing the biotechnological potential of pAgo proteins.
History
DepositionMay 28, 2025Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 15, 2026Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 15, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: VbAgo
B: DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T)-3')
C: RNA (5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*A)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)100,1046
Polymers100,0323
Non-polymers733
Water25214
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 VbAgo


Mass: 88463.320 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Verrucomicrobiota bacterium (bacteria) / Production host: Escherichia coli (E. coli)
#2: DNA chain DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T)-3')


Mass: 5641.660 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Verrucomicrobiota bacterium (bacteria)
#3: RNA chain RNA (5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*A)-3')


Mass: 5926.584 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Verrucomicrobiota bacterium (bacteria)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 14 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: VbAgo ternary complex / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Verrucomicrobiota 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: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1500 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOCONTINUUM (6k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.19.2_4158model refinement
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 112179 / Symmetry type: POINT
RefinementHighest resolution: 2.97 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0036711
ELECTRON MICROSCOPYf_angle_d0.4919208
ELECTRON MICROSCOPYf_dihedral_angle_d12.511091
ELECTRON MICROSCOPYf_chiral_restr0.041021
ELECTRON MICROSCOPYf_plane_restr0.0051128

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