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- PDB-9jhm: Cryo-EM structure of CpAgo_gDNA-tg_bubble_dsDNA monomeric ternary... -

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Basic information

Entry
Database: PDB / ID: 9jhm
TitleCryo-EM structure of CpAgo_gDNA-tg_bubble_dsDNA monomeric ternary complex
Components
  • Clostridium perfringen Argonaute
  • DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*TP*AP*GP*T)-3')
  • DNA (58-MER)
KeywordsDNA BINDING PROTEIN/DNA / pAgos / nucleotide-binding pocket / PAZ / cryo-EM / DNA BINDING PROTEIN-DNA complex
Function / homology: / DNA / DNA (> 10)
Function and homology information
Biological speciesClostridium perfringens (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLiu, Y. / Li, S. / Zhang, K.
Funding support1items
OrganizationGrant numberCountry
Other government
CitationJournal: Nat Commun / Year: 2025
Title: The PAZ pocket and dimerization drive CpAgo's guide-independent and DNA-guided dual catalysis.
Authors: Yuchan Liu / Jiasu Zhang / Ji Liu / Shengchun Zhang / Linfeng An / Wenbing Xie / Kaiming Zhang / Shanshan Li /
Abstract: Argonaute proteins (Agos) play essential roles in nucleic acid targeting across life domains. While eukaryotic Agos (eAgos) utilize small-interfering RNAs (siRNAs) or microRNAs (miRNAs) for RNA ...Argonaute proteins (Agos) play essential roles in nucleic acid targeting across life domains. While eukaryotic Agos (eAgos) utilize small-interfering RNAs (siRNAs) or microRNAs (miRNAs) for RNA interference, the mechanisms driving prokaryotic Agos (pAgos) in bacterial defense remain underexplored. Here, we characterize the mesophilic pAgo from Clostridium perfringens (CpAgo), which exhibits robust guide-independent and DNA-guided activity at 37 °C. CpAgo efficiently degrades plasmids and structured RNAs into small fragments, generating DNA fragments that serve as guides for subsequent cleavage. Cryo-electron microscopy reveals a positively-charged PAZ nucleotide-binding pocket, critical for both guide-dependent and guide-independent substrate recognition and cleavage. Structural analysis identifies CpAgo's dimerization as a prerequisite for catalytic activity, supporting both nucleic acid degradation and targeted action. Functional assays in Escherichia coli demonstrate CpAgo's role in bacterial defense by mediating plasmid degradation and DNA-guided cleavage. These findings position CpAgo as a critical component of prokaryotic immunity and a promising tool for biotechnology.
History
DepositionSep 10, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jul 30, 2025Provider: repository / Type: Initial release
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Clostridium perfringen Argonaute
C: DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*TP*AP*GP*T)-3')
E: DNA (58-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)110,6504
Polymers110,5953
Non-polymers551
Water00
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 Clostridium perfringen Argonaute


Mass: 86285.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Clostridium perfringens (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*TP*AP*GP*T)-3')


Mass: 6588.266 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (58-MER)


Mass: 17721.516 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
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: Cryo-EM structure of CpAgo_gDNA-tg_bubble_dsDNA monomeric ternary complex
Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.1 MDa / Experimental value: YES
Source (natural)Organism: Clostridium perfringens (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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: 3000 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 48.6 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2EPUimage acquisition
8PHENIXmodel refinement
12cryoSPARC4.5.1classification
13cryoSPARC4.5.13D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 1609554
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 159745 / Symmetry type: POINT
RefinementStereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0066963
ELECTRON MICROSCOPYf_angle_d0.9589536
ELECTRON MICROSCOPYf_dihedral_angle_d10.7174092
ELECTRON MICROSCOPYf_chiral_restr0.0611044
ELECTRON MICROSCOPYf_plane_restr0.0061090

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