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- EMDB-61780: inactive NbaSPARDA complexes -

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Basic information

Entry
Database: EMDB / ID: EMD-61780
Titleinactive NbaSPARDA complexes
Map data
Sample
  • Complex: pro-RNA-13DNA
    • Protein or peptide: Ago
    • Protein or peptide: DREN-APAZ
    • RNA: RNA (5'-R(P*AP*UP*AP*CP*UP*GP*CP*AP*CP*AP*GP*CP*UP*GP*AP*CP*GP*AP*UP*A)-3')
    • DNA: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*CP*AP*GP*TP*AP*TP*T)-3')
  • Ligand: MAGNESIUM ION
Keywordsprotein / RNA / DNA / RNA BINDING PROTEIN/RNA/DNA / RNA BINDING PROTEIN-RNA-DNA complex
Biological speciesNovosphingopyxis baekryungensis DSM 16222 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsZhuang L
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Res / Year: 2025
Title: Target DNA-induced filament formation and nuclease activation of SPARDA complex.
Authors: Feng Wang / Haijiang Xu / Chendi Zhang / Jialin Xue / Zhuang Li /
Abstract: The short Argonaute-based bacterial defense system, SPARDA (Short Prokaryotic Argonaute and DNase/RNase-APAZ), utilizes guide RNA to target invading complementary DNA and exhibits collateral nuclease ...The short Argonaute-based bacterial defense system, SPARDA (Short Prokaryotic Argonaute and DNase/RNase-APAZ), utilizes guide RNA to target invading complementary DNA and exhibits collateral nuclease activity, leading to cell death or dormancy. However, its detailed mechanisms remain poorly understood. In this study, we investigated the SPARDA system from Novosphingopyxis baekryungensis (NbaSPARDA) and discovered an unexpected filament configuration upon target DNA binding, which strongly correlated with collateral nuclease activity. Filament formation and nuclease activation require a guide-target heteroduplex of sufficient length with perfect complementarity at the central region. A series of cryo-EM structures of NbaSPARDA complexes, loaded with guide RNA, target DNA of varying lengths, and substrate ssDNA, were determined at ~3.0 Å resolution. Structural analyses indicated that guide RNA binding induces dimerization of the NbaSPARDA complex, while target DNA engagement disrupts this dimerization. Further propagation of the guide-target heteroduplex triggers filament formation through a checkpoint mechanism. The NbaSPARDA filament consists of a backbone formed by interlocking short Argonaute proteins, with an inner layer composed of DREN nuclease domains. Filament formation leads to tetramerization of the monomeric DREN nuclease domain, activating its collateral nuclease activity against environmental nucleic acids - a feature leveraged for molecular diagnostics. For bacteria heterologously expressing the NbaSPARDA system, defense against invading bacteriophages and plasmids relies on filament formation. Collectively, these findings illustrate the detailed working mechanism of the NbaSPARDA complex and highlight the importance of its filament formation in host defense.
History
DepositionSep 30, 2024-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateApr 2, 2025-
Current statusApr 2, 2025Processing site: PDBc / Status: Released

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Structure visualization

Supplemental images

emd_61780.png

Downloads & links

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Map

FileDownload / File: emd_61780.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.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å		0.85 Å/pix.
x 256 pix.
= 217.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.85 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.258
Minimum - Maximum-3.2208674 - 4.049473
Average (Standard dev.)-0.00087377673 (±0.061530426)
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_61780_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_61780_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 : pro-RNA-13DNA

EntireName: pro-RNA-13DNA
Components
  • Complex: pro-RNA-13DNA
    • Protein or peptide: Ago
    • Protein or peptide: DREN-APAZ
    • RNA: RNA (5'-R(P*AP*UP*AP*CP*UP*GP*CP*AP*CP*AP*GP*CP*UP*GP*AP*CP*GP*AP*UP*A)-3')
    • DNA: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*CP*AP*GP*TP*AP*TP*T)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: pro-RNA-13DNA

SupramoleculeName: pro-RNA-13DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Novosphingopyxis baekryungensis DSM 16222 (bacteria)

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Macromolecule #1: Ago

MacromoleculeName: Ago / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Molecular weightTheoretical: 54.492809 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTFETRIFDE PELEFGDHHH HQDPRLGLSE AGPLQTFLGD VIKIGVVGNS KTIEDTRKFI ETVSSGVEGK GEKHPNMHPP FPGLGNQSP YRCRFEIEDG ATAALTKSKL DKIGKEPDHY RAVEMAVDEI IGELQAMDDG GSRPDVAIIA LPVKLLERVW N AKVDARGT ...String:
MTFETRIFDE PELEFGDHHH HQDPRLGLSE AGPLQTFLGD VIKIGVVGNS KTIEDTRKFI ETVSSGVEGK GEKHPNMHPP FPGLGNQSP YRCRFEIEDG ATAALTKSKL DKIGKEPDHY RAVEMAVDEI IGELQAMDDG GSRPDVAIIA LPVKLLERVW N AKVDARGT TEKSDSSGSD APNFRGMLKA KAMGLSFPIQ IVWEDVIDDK VTIPQKVKES SSRKIQDIAG RTWNLMTSLY YK GSGRIPW RRMPLEGEFS ACYVGISFYR EADGQQLFTS AAQMFDERGR GFVLKGRRAR TESRGRHPYM AREDAKKIIE DVL AAYKLH HKTLPARVFI LKTSRFKDEE ADGIIAALDE AGTELRDLVW VQESYTARIL RDGNYPVLRG TFVDLHGKGL LYTS GSMPY YGTYPGKYDP NPLLLCPHHT SESTVAQLAE EIFSLTKVNW NSTQMNQRLP IPIRAARKVG EVLKYVGEGE VISAD YRKY I

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Macromolecule #2: DREN-APAZ

MacromoleculeName: DREN-APAZ / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Molecular weightTheoretical: 50.419645 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MTKKITANQI IGEIGENEVR GRFLTLGWQF DGRSRLEAGI DGIAEVMNEG QPMARMIAVQ IKSTKEGKYT SESDTSFTYL LRTQDLAYW RGSNLPVIVV FYRQSDHSFY WKEVSRDAGP GERRLNIDKV ADLFNASTVN KLAALTVPKT GLGYYVPPLG G GEDALINM ...String:
MTKKITANQI IGEIGENEVR GRFLTLGWQF DGRSRLEAGI DGIAEVMNEG QPMARMIAVQ IKSTKEGKYT SESDTSFTYL LRTQDLAYW RGSNLPVIVV FYRQSDHSFY WKEVSRDAGP GERRLNIDKV ADLFNASTVN KLAALTVPKT GLGYYVPPLG G GEDALINM LPLTLPNEMY IASTTYEPRK AIAVILNGDG PKRFDWVING GTFWSFHDPR TSACSEIVDI DQVEAINTKE LA LHDDIDE QNRFSHLLRQ TLRYQTDSDL GWDKDHKALY FRAIEREVSR NFAYTSSKKK TDANVVSVFK NSKDETRVSF VRH HAFSPR FELMADQWYL IITPTYYYTT NGYAPHQFAA PLLAGKKRLD KSAALRGQVI MWHRFLTQSD HEDLFHSEET PEAY LMFGE PPSIHLDVRV PEDGWVKEKV KRIDEAAQGE GLFSDDI

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

MacromoleculeName: RNA (5'-R(P*AP*UP*AP*CP*UP*GP*CP*AP*CP*AP*GP*CP*UP*GP*AP*CP*GP*AP*UP*A)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Molecular weightTheoretical: 6.390879 KDa
SequenceString:
AUACUGCACA GCUGACGAUA

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Macromolecule #4: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*CP*AP*GP*TP*AP*TP*T)-3')

MacromoleculeName: DNA (5'-D(P*GP*CP*TP*GP*TP*GP*CP*AP*GP*TP*AP*TP*T)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Novosphingopyxis baekryungensis DSM 16222 (bacteria)
Molecular weightTheoretical: 3.997607 KDa
SequenceString:
(DG)(DC)(DT)(DG)(DT)(DG)(DC)(DA)(DG)(DT) (DA)(DT)(DT)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / 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 TALOS ARCTICA
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: 2.2 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 213852
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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