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- EMDB-36489: Cryo-EM structure of PfAgo-guide DNA-target DNA complex -

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Basic information

Entry
Database: EMDB / ID: EMD-36489
TitleCryo-EM structure of PfAgo-guide DNA-target DNA complex
Map data
Sample
  • Complex: Cryo-EM structure of a PfAgo-guide DNA-target DNA complex
    • Protein or peptide: Protein argonaute
    • DNA: Guide DNA
    • DNA: Target DNA
    • DNA: Excess DNA
  • Ligand: MAGNESIUM ION
Keywordsnuclease / GENE REGULATION
Function / homology
Function and homology information


Hydrolases; Acting on ester bonds; Site specific endodeoxyribonucleases: cleavage is not sequence specific (deleted sub-subclass) / clearance of foreign intracellular DNA / DNA endonuclease activity / manganese ion binding / DNA binding
Similarity search - Function
Argonaute PAZ domain, archaea / PAZ domain / Piwi domain / Piwi domain profile. / Piwi domain / Piwi / PAZ domain superfamily / PAZ domain profile. / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Biological speciesPyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1) (archaea) / Pyrococcus furiosus DSM 3638 (archaea)
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
DepositionJun 13, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_36489.png

Downloads & links

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Map

FileDownload / File: emd_36489.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.85 Å
Density
Contour LevelBy AUTHOR: 0.45
Minimum - Maximum-1.0716412 - 2.6407375
Average (Standard dev.)0.007128707 (±0.11640368)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 170.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_36489_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_36489_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 a PfAgo-guide DNA-target DNA complex

EntireName: Cryo-EM structure of a PfAgo-guide DNA-target DNA complex
Components
  • Complex: Cryo-EM structure of a PfAgo-guide DNA-target DNA complex
    • Protein or peptide: Protein argonaute
    • DNA: Guide DNA
    • DNA: Target DNA
    • DNA: Excess DNA
  • Ligand: MAGNESIUM ION

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

SupramoleculeName: Cryo-EM structure of a PfAgo-guide DNA-target DNA complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1) (archaea)

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Macromolecule #1: Protein argonaute

MacromoleculeName: Protein argonaute / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Site specific endodeoxyribonucleases: cleavage is not sequence specific (deleted sub-subclass)
Source (natural)Organism: Pyrococcus furiosus (strain ATCC 43587 / DSM 3638 / JCM 8422 / Vc1) (archaea)
Molecular weightTheoretical: 90.510094 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKAIVVINLV KINKKIIPDK IYVYRLFNDP EEELQKEGYS IYRLAYENVG IVIDPENLII ATTKELEYEG EFIPEGEISF SELRNDYQS KLVLRLLKEN GIGEYELSKL LRKFRKPKTF GDYKVIPSVE MSVIKHDEDF YLVIHIIHQI QSMKTLWELV N KDPKELEE ...String:
MKAIVVINLV KINKKIIPDK IYVYRLFNDP EEELQKEGYS IYRLAYENVG IVIDPENLII ATTKELEYEG EFIPEGEISF SELRNDYQS KLVLRLLKEN GIGEYELSKL LRKFRKPKTF GDYKVIPSVE MSVIKHDEDF YLVIHIIHQI QSMKTLWELV N KDPKELEE FLMTHKENLM LKDIASPLKT VYKPCFEEYT KKPKLDHNQE IVKYWYNYHI ERYWNTPEAK LEFYRKFGQV DL KQPAILA KFASKIKKNK NYKIYLLPQL VVPTYNAEQL ESDVAKEILE YTKLMPEERK ELLENILAEV DSDIIDKSLS EIE VEKIAQ ELENKIRVRD DKGNSVPISQ LNVQKSQLLL WTNYSRKYPV ILPYEVPEKF RKIREIPMFI ILDSGLLADI QNFA TNEFR ELVKSMYYSL AKKYNSLAKK ARSTNEIGLP FLDFRGKEKV ITEDLNSDKG IIEVVEQVSS FMKGKELGLA FIAAR NKLS SEKFEEIKRR LFNLNVISQV VNEDTLKNKR DKYDRNRLDL FVRHNLLFQV LSKLGVKYYV LDYRFNYDYI IGIDVA PMK RSEGYIGGSA VMFDSQGYIR KIVPIKIGEQ RGESVDMNEF FKEMVDKFKE FNIKLDNKKI LLLRDGRITN NEEEGLK YI SEMFDIEVVT MDVIKNHPVR AFANMKMYFN LGGAIYLIPH KLKQAKGTPI PIKLAKKRII KNGKVEKQSI TRQDVLDI F ILTRLNYGSI SADMRLPAPV HYAHKFANAI RNEWKIKEEF LAEGFLYFV

UniProtKB: Protein argonaute

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Macromolecule #2: Guide DNA

MacromoleculeName: Guide DNA / type: dna / ID: 2 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 5.337467 KDa
SequenceString:
(DT)(DG)(DA)(DG)(DG)(DT)(DA)(DG)(DT)(DA) (DG)(DG)(DT)(DT)(DG)(DT)(DA)

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Macromolecule #3: Target DNA

MacromoleculeName: Target DNA / type: dna / ID: 3 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 5.075327 KDa
SequenceString:
(DA)(DC)(DA)(DA)(DC)(DC)(DT)(DA)(DC)(DT) (DA)(DC)(DC)(DT)(DC)(DA)(DT)

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Macromolecule #4: Excess DNA

MacromoleculeName: Excess DNA / type: dna / ID: 4 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: Pyrococcus furiosus DSM 3638 (archaea)
Molecular weightTheoretical: 1.780199 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)

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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: 52.0 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: 621102
FSC plot (resolution estimation)

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