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- EMDB-37457: Cryo-EM structure of TtdAgo-guide DNA-target DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-37457
TitleCryo-EM structure of TtdAgo-guide DNA-target DNA complex
Map data
Sample
  • Complex: Cryo-EM structure of TtdAgo-guide DNA-target DNA complex
    • Complex: DNA binding proteinDNA-binding protein
      • Protein or peptide: Argonaute family protein
    • Complex: DNA
      • DNA: Target DNA
      • DNA: Guide DNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION
KeywordsDNA 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
Argonaute family protein
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsZhuang L
Funding support1 items
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, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37457.png

Downloads & links

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Map

FileDownload / File: emd_37457.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-2.3344398 - 4.168501
Average (Standard dev.)0.0005319172 (±0.14511278)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 217.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_37457_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_37457_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of TtdAgo-guide DNA-target DNA complex

EntireName: Cryo-EM structure of TtdAgo-guide DNA-target DNA complex
Components
  • Complex: Cryo-EM structure of TtdAgo-guide DNA-target DNA complex
    • Complex: DNA binding proteinDNA-binding protein
      • Protein or peptide: Argonaute family protein
    • Complex: DNA
      • DNA: Target DNA
      • DNA: Guide DNA
  • Ligand: ZINC ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Cryo-EM structure of TtdAgo-guide DNA-target DNA complex

SupramoleculeName: Cryo-EM structure of TtdAgo-guide DNA-target DNA complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Thermus thermophilus (bacteria)

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Supramolecule #2: DNA binding protein

SupramoleculeName: DNA binding protein / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1

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Supramolecule #3: DNA

SupramoleculeName: DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3

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Macromolecule #1: Argonaute family protein

MacromoleculeName: Argonaute family protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 87.488398 KDa
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString: MLMKVLTNMV KLNQDIIPNE IYLYKIFNKP EDGMNIYKIA YRNHGIVIDP QNRIIATPSE LEYSGKFAIE DEISFNELPE NYQNRLVLR ILRDNGISDH ALSRTLQKYR KPKPFGDFEV IPEIRSSVIK HGGDFYLVLH LSHQIRSKKT LWELVGRNKD A LRDFLKEH ...String:
MLMKVLTNMV KLNQDIIPNE IYLYKIFNKP EDGMNIYKIA YRNHGIVIDP QNRIIATPSE LEYSGKFAIE DEISFNELPE NYQNRLVLR ILRDNGISDH ALSRTLQKYR KPKPFGDFEV IPEIRSSVIK HGGDFYLVLH LSHQIRSKKT LWELVGRNKD A LRDFLKEH RGTILLRDIA SEHKVVYKPI FKRYNGDPDL IEDNSNDVEH WYDYHLERYW NTPELKKEFY KKFGPVDLNQ PI ILAKPLR QHNRGDLVHL LPQFVVPVYN AEQLNDILAS EILEYLKLTS NQRISLLSRL INDIKTNTNI IVSSLTELEA NTF DVDLND MLQVRNADNV KVTLSELEIS KTRLFTWMKS RKYPVILPYD IPQKLKKIEK IPVFIIVDSA LSRDIQTFAK DEFR YLISS LQKSLSNWVD FPILDIRDKY IFTIDLTSDK DIVNLSIKLV NLMKNAELGL ALIATRTKLP NETFDEVKKR LFSVN IISQ VVNEATLYKR DKYNESRLNL YVQHNLLFQI LSKLGIKYYV LRHKFSYDYI VGIDVTPMKL SHGYIGGSAV MFDSQG YIR KIIPVEIGEQ MGESIDMKEF FKDMVVQFGK FGIDLEGKSI LILRDGKITK DEEEGLAYIS KVFGIKITTF NIVKRHL LR IFANRKLYLR LANSVYLLPH RIKQSVGTPV PLKLSEKRLI LDGTITSQEI TYNDIFEILL LSELNYGSIS ADMKLPAP V HYAHKFVRAL RKGWRIREEL LAEGFLYFV

UniProtKB: Argonaute family protein

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Macromolecule #2: Target DNA

MacromoleculeName: Target DNA / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 5.051302 KDa
SequenceString:
(DA)(DC)(DA)(DA)(DC)(DC)(DT)(DA)(DC)(DT) (DA)(DC)(DC)(DT)(DC)(DC)(DT)

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Macromolecule #3: Guide DNA

MacromoleculeName: Guide DNA / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Thermus thermophilus (bacteria)
Molecular weightTheoretical: 5.049273 KDa
SequenceString:
(DG)(DG)(DA)(DG)(DG)(DT)(DA)(DG)(DT)(DA) (DG)(DG)(DT)(DT)(DG)(DT)

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.2 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 629194
FSC plot (resolution estimation)

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