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- EMDB-36946: Dimer structure of procaryotic Ago -

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Basic information

Entry
Database: EMDB / ID: EMD-36946
TitleDimer structure of procaryotic Ago
Map datacombined focused map, sharpened with deepemhancer
Sample
  • Complex: dimmeric GsSirt2/ago-gRAN-tDNA complex
    • DNA: DNA (41-mer)
    • Other: RNA/DNA (21-mer)
    • Protein or peptide: Piwi domain protein
    • Protein or peptide: Sir2 superfamily protein
  • Ligand: MAGNESIUM ION
KeywordsAgo / DNA/RNA / DNA BINDING PROTEIN / CELL INVASION / DNA-RNA-CELL INVASION complex
Function / homologySIR2-like domain / SIR2-like domain / DHS-like NAD/FAD-binding domain superfamily / Ribonuclease H superfamily / nucleic acid binding / Ribonuclease H-like superfamily / Piwi domain protein / Sir2 superfamily protein
Function and homology information
Biological speciesGeobacter sulfurreducens (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsGao X / Sun D / Cui S / Wang Y
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Rep / Year: 2024
Title: Nucleic acid-induced NADase activation of a short Sir2-associated prokaryotic Argonaute system.
Authors: Dapeng Sun / Kaixiang Zhu / Linyue Wang / Zhixia Mu / Kang Wu / Lei Hua / Bo Qin / Xiaopan Gao / Yumei Wang / Sheng Cui /
Abstract: Inhibition of nucleic acid targets is mediated by Argonaute (Ago) proteins guided by RNA or DNA. Although the mechanisms underpinning the functions of eukaryotic and "long" prokaryotic Ago proteins ...Inhibition of nucleic acid targets is mediated by Argonaute (Ago) proteins guided by RNA or DNA. Although the mechanisms underpinning the functions of eukaryotic and "long" prokaryotic Ago proteins (pAgos) are well understood, those for short pAgos remain enigmatic. Here, we determine two cryoelectron microscopy structures of short pAgos in association with the NADase-domain-containing protein Sir2-APAZ from Geobacter sulfurreducens (GsSir2/Ago): the guide RNA-target DNA-loaded GsSir2/Ago quaternary complex (2.58 Å) and the dimer of the quaternary complex (2.93Å). These structures show that the nucleic acid binding causes profound conformational changes that result in disorder or partial dissociation of the Sir2 domain, suggesting that it adopts a NADase-active conformation. Subsequently, two RNA-/DNA-loaded GsSir2/Ago complexes form a dimer through their MID domains, further enhancing NADase activity through synergistic effects. The findings provide a structural basis for short-pAgo-mediated defense against invading nucleic acids.
History
DepositionJul 29, 2023-
Header (metadata) releaseJul 3, 2024-
Map releaseJul 3, 2024-
UpdateSep 18, 2024-
Current statusSep 18, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_36946.png

Downloads & links

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Map

FileDownload / File: emd_36946.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationcombined focused map, sharpened with deepemhancer
Projections & slices

Image control

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AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 256 pix.
= 258.304 Å		1.01 Å/pix.
x 256 pix.
= 258.304 Å		1.01 Å/pix.
x 256 pix.
= 258.304 Å

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.009 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.00086645293 - 2.4011953
Average (Standard dev.)0.0050141406 (±0.057758145)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 258.304 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_36946_msk_1.map
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Half map: #2

Fileemd_36946_half_map_1.map
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Half map: #1

Fileemd_36946_half_map_2.map
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Sample components

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Entire : dimmeric GsSirt2/ago-gRAN-tDNA complex

EntireName: dimmeric GsSirt2/ago-gRAN-tDNA complex
Components
  • Complex: dimmeric GsSirt2/ago-gRAN-tDNA complex
    • DNA: DNA (41-mer)
    • Other: RNA/DNA (21-mer)
    • Protein or peptide: Piwi domain protein
    • Protein or peptide: Sir2 superfamily protein
  • Ligand: MAGNESIUM ION

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Supramolecule #1: dimmeric GsSirt2/ago-gRAN-tDNA complex

SupramoleculeName: dimmeric GsSirt2/ago-gRAN-tDNA complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Geobacter sulfurreducens (bacteria)

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Macromolecule #1: DNA (41-mer)

MacromoleculeName: DNA (41-mer) / type: dna / ID: 1 / Number of copies: 2 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 12.525115 KDa
SequenceString:
(DC)(DG)(DA)(DA)(DA)(DC)(DA)(DA)(DC)(DA) (DC)(DA)(DG)(DC)(DT)(DC)(DT)(DA)(DA)(DC) (DC)(DG)(DA)(DC)(DG)(DT)(DC)(DT)(DA) (DA)(DG)(DA)(DA)(DA)(DC)(DC)(DA)(DT)(DT) (DA) (DT)

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Macromolecule #2: RNA/DNA (21-mer)

MacromoleculeName: RNA/DNA (21-mer) / type: other / ID: 2 / Number of copies: 2
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 6.675973 KDa
SequenceString:
AUAAUGGUUU CUUAGACGUC (DG)

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Macromolecule #3: Piwi domain protein

MacromoleculeName: Piwi domain protein / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightTheoretical: 53.325566 KDa
Recombinant expressionOrganism: Expression vector pET-mod (others)
SequenceString: MADNLSQLAA HSTIPEPLLL FKDNRTDTHP LRGLSQYGPY SACFNLPGQV RLAYLAPTEH MRKLDAIVRE LQNPATPKEA TNYYVEYGG FEKVFKVPLV MPQEHLRCLA LDECHGVAAN GNGLALADKI VQSMSGLFRQ KHAFDVLLVY LPASWKKCFE Y DGFDLHDR ...String:
MADNLSQLAA HSTIPEPLLL FKDNRTDTHP LRGLSQYGPY SACFNLPGQV RLAYLAPTEH MRKLDAIVRE LQNPATPKEA TNYYVEYGG FEKVFKVPLV MPQEHLRCLA LDECHGVAAN GNGLALADKI VQSMSGLFRQ KHAFDVLLVY LPASWKKCFE Y DGFDLHDR IKAKVAPLNL PIQIINDTAL TRQCRANVMW GVSVALYAKA GGIPWKLADW DKDEAYIGLS YAIKKNAEGQ EY TTCCSQV FDPDGTGFEF VAYDTREFIT DRKGNPYLSY QEMQSVLSKS LHLYQSSHNG RMPRKIFIHK TTHFTEDEIQ GAF DSFSSS TEIELVQIIQ STNWYGLKVD GKKGDKPVAP ASYPVDRGLY QPLTESECLL WTQGSVMGVN QQNPGQPVFK EAAL TPLPN PIMLRRFSGN GGWHATCSSI LALTKVDWNN NTLYKKLPVT LVYSQVFADV VKQTPEIVNE IYDYRFFM

UniProtKB: Piwi domain protein

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Macromolecule #4: Sir2 superfamily protein

MacromoleculeName: Sir2 superfamily protein / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Geobacter sulfurreducens (bacteria)
Molecular weightTheoretical: 66.645922 KDa
Recombinant expressionOrganism: Expression vector pET-mod (others)
SequenceString: MDVLTDNEFY QHYLQNSQHM MWFLGAGTSR SAGLPTASDI IWDLKHRYYC LHENQDYQKH DINNHAIKSK IQSYMDSKGF PLQWSPEEY SFYFELVFRD DYEAQRKYLL EALASRKVSL NIGHRVLAAL LEMNQTKVVF TTNFDDVIET AFSDISGKHL S VYHLEGSY ...String:
MDVLTDNEFY QHYLQNSQHM MWFLGAGTSR SAGLPTASDI IWDLKHRYYC LHENQDYQKH DINNHAIKSK IQSYMDSKGF PLQWSPEEY SFYFELVFRD DYEAQRKYLL EALASRKVSL NIGHRVLAAL LEMNQTKVVF TTNFDDVIET AFSDISGKHL S VYHLEGSY AALSALNTEA FPIYAKIHGD FRYQKIKNLT PDLQTNDREI HKCFLAAAIR FGLVVSGYSG RDENVMTMLR AA IDQNNAF PHGLYWTVPS ISKSEPAVQD LITYAQGKGV RAYLVETGTF DEMLSKIWRQ VKDKPAAIDA KVRTARVCPV SIP LPGPGK SFPALRTNAL PVVTQSIRCG VVTLASPITF SELKERISQK SPKALLTYTE KVLFLGGEPE IRKIFSNDEI NSIG QYYID EIAQSVAAST FLKSFVEEAI LTALLREKPI LHRVRHRTHY AVIPNASAKD DRFLDLRKAV GFKGDLGYIT GNVTN AKEL SWAEAVSIRL EERGGKLWIM LKPEIWIKPL DRREEATDFI RSRRRYRFNQ CSYQILDAWI KILFGSIGGG GTVNIS CFP DAEFKAEFEI GTRTAFSLGV GYGR

UniProtKB: Sir2 superfamily protein

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 2 / 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

Concentration1 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: cryosparc ab-initio reconstruction
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 1103892
Initial angle assignmentType: OTHER
Final angle assignmentType: MAXIMUM LIKELIHOOD

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