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- EMDB-64873: The transition-state structure of the Argonaute protein from a Ve... -

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Basic information

Entry
Database: EMDB / ID: EMD-64873
TitleThe transition-state structure of the Argonaute protein from a Verrucomicrobia bacterium in complex with guide DNA and target RNA.
Map data
Sample
  • Complex: VbAgo ternary complex
    • Protein or peptide: VbAgo
    • DNA: DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T)-3')
    • RNA: RNA (5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*A)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water
Keywordsmesophilic prokaryotic Argonaute / RNA BINDING PROTEIN/DNA/RNA / RNA BINDING PROTEIN-DNA-RNA complex
Biological speciesVerrucomicrobiota bacterium (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 Å
AuthorsWu S
Funding support China, 1 items
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 30, 2025-
Header (metadata) releaseApr 15, 2026-
Map releaseApr 15, 2026-
UpdateApr 15, 2026-
Current statusApr 15, 2026Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_64873.png

Downloads & links

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Map

FileDownload / File: emd_64873.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 256 pix.
= 217.856 Å		0.85 Å/pix.
x 256 pix.
= 217.856 Å		0.85 Å/pix.
x 256 pix.
= 217.856 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.851 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-1.1990519 - 2.023914
Average (Standard dev.)-0.00027792685 (±0.035152692)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.856 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_64873_half_map_1.map
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_64873_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : VbAgo ternary complex

EntireName: VbAgo ternary complex
Components
  • Complex: VbAgo ternary complex
    • Protein or peptide: VbAgo
    • DNA: DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T)-3')
    • RNA: RNA (5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*A)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: VbAgo ternary complex

SupramoleculeName: VbAgo ternary complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Verrucomicrobiota bacterium (bacteria)

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Macromolecule #1: VbAgo

MacromoleculeName: VbAgo / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Verrucomicrobiota bacterium (bacteria)
Molecular weightTheoretical: 88.46332 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSEKQLGATL FPITGLPAQA FRLRVLRVRE TIPMDTQTPV RLNRWATQLW KELKQAVVPT GRFEWPAFLT PDVESLTVGR VLTVQDVPD REYSIEVIGE TVEVNPASAS SEELQLAGEM IKRAISDAFG RNSDKYWRKH WNLYFRLEPE NLQDRRDRVF A YRGLKFSV ...String:
MSEKQLGATL FPITGLPAQA FRLRVLRVRE TIPMDTQTPV RLNRWATQLW KELKQAVVPT GRFEWPAFLT PDVESLTVGR VLTVQDVPD REYSIEVIGE TVEVNPASAS SEELQLAGEM IKRAISDAFG RNSDKYWRKH WNLYFRLEPE NLQDRRDRVF A YRGLKFSV VFLGDKPWLA ADILTTYHGQ HALSEYSSEQ RQRELHFHVS ERIEADDRAM FLRDNGKIKI PCRFVGSTGK TV TQYTFPI NGGQKNVREY YEQRYGIRVP ENDEAVFVRD REGCDSWPVP ASRLFPLFTT EYDEVRNCSV VPQMPPDERV ETI RAFLND LRDVSFAGST LAIGHSHFQT AERSVFPAPA LEFGNGQTLT VDASLPIEEG YNRYRQGKMT MLYEHGPFSS QSLP DLVLL YPDNLDRNAR EKLRQRLGEE IKELCGVAPR IARQISYPLG KQPHAGAGLL AAADELVRNN DGTFLPVIVL ADALR EHIY DLLKRRLSSL ASQCVRERTV ARVARDEQAV GGSRLRNLAL GILTAAGLQP WVLAKPLHYD FYMGVDLLAN QVIYVF VCG KGGRNVWVQR GDQLRRRGIT EKIDRVQLAD QFKTGVREAK RLGVPLNSLV VHRDGRWWSN EDLAITEAVA ELQGDGT LS KDCQVGVVEV RKSHLPVRLF SVLNATKGSL ENPMPGSHLI LNNTEAILTP TGQPGRWDKQ GRTAGTLLLR ITRNPNGS P LDIRKIAEDA YGLTHLNWNA PDIEISLPVT IRWSDERLRE IVTNPSATDD TEVEPQETCI V

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Macromolecule #2: DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T)-3')

MacromoleculeName: DNA (5'-D(P*TP*GP*AP*GP*GP*TP*AP*GP*TP*AP*GP*GP*TP*TP*GP*TP*AP*T)-3')
type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Verrucomicrobiota bacterium (bacteria)
Molecular weightTheoretical: 5.64166 KDa
SequenceString:
(DT)(DG)(DA)(DG)(DG)(DT)(DA)(DG)(DT)(DA) (DG)(DG)(DT)(DT)(DG)(DT)(DA)(DT)

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Macromolecule #3: RNA (5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*...

MacromoleculeName: RNA (5'-R(*UP*AP*UP*AP*CP*AP*AP*CP*CP*UP*AP*CP*UP*AP*CP*CP*UP*CP*A)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Verrucomicrobiota bacterium (bacteria)
Molecular weightTheoretical: 5.926584 KDa
SequenceString:
UAUACAACCU ACUACCUCA

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 8 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

9v65

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 114582
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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