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- PDB-9qcc: Activated XauSPARDA filament assembly with bound dsDNA substrate -

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Basic information

Entry
Database: PDB / ID: 9qcc
TitleActivated XauSPARDA filament assembly with bound dsDNA substrate
Components
  • (dsDNA substrate, ...) x 2
  • DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
  • DUF4365 domain-containing protein
  • Protein argonaute
  • RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
KeywordsRNA BINDING PROTEIN / short prokaryotic Argonaute / bacterial defence system / DREN domain / DREN-APAZ
Function / homology
Function and homology information


nucleic acid binding
Similarity search - Function
Domain of unknown function DUF4365 / Domain of unknown function (DUF4365) / Piwi domain / Piwi / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / DUF4365 domain-containing protein / Protein argonaute
Similarity search - Component
Biological speciesXanthobacter autotrophicus Py2 (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsManakova, E.N. / Zaremba, M. / Jurgelaitis, E.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Res / Year: 2025
Title: Activation of the SPARDA defense system by filament assembly using a beta-relay signaling mechanism widespread in prokaryotic Argonautes.
Authors: Edvinas Jurgelaitis / Evelina Zagorskaitė / Aurimas Kopūstas / Simonas Asmontas / Elena Manakova / Indrė Dalgėdienė / Ugnė Tylenytė / Arunas Silanskas / Paulius Toliusis / Algirdas ...Authors: Edvinas Jurgelaitis / Evelina Zagorskaitė / Aurimas Kopūstas / Simonas Asmontas / Elena Manakova / Indrė Dalgėdienė / Ugnė Tylenytė / Arunas Silanskas / Paulius Toliusis / Algirdas Grybauskas / Marijonas Tutkus / Česlovas Venclovas / Mindaugas Zaremba
Abstract: Present in all three domains of life, Argonaute proteins use short oligonucleotides as guides to recognize complementary nucleic acid targets. In eukaryotes, Argonautes are involved in RNA silencing, ...Present in all three domains of life, Argonaute proteins use short oligonucleotides as guides to recognize complementary nucleic acid targets. In eukaryotes, Argonautes are involved in RNA silencing, whereas in prokaryotes, they function in host defense against invading DNA. Here, we show that SPARDA (short prokaryotic Argonaute, DNase associated) systems from Xanthobacter autotrophicus (Xau) and Enhydrobacter aerosaccus (Eae) function in anti-plasmid defense. Upon activation, SPARDA nonspecifically degrades both invader and genomic DNA, causing host death, thereby preventing further spread of the invader in the population. X-ray structures of the apo Xau and EaeSPARDA complexes show that they are dimers, unlike other apo short pAgo systems, which are monomers. We show that dimerization in the apo state is essential for inhibition of XauSPARDA activity. We demonstrate by cryo-EM that activated XauSPARDA forms a filament. Upon activation, the recognition signal of the bound guide/target duplex is relayed to other functional XauSPARDA sites through a structural region that we termed the "beta-relay". Owing to dramatic conformational changes associated with guide/target binding, XauSPARDA undergoes a "dimer-monomer-filament" transition as the apo dimer dissociates into the guide/target-loaded monomers that subsequently assemble into the filament. Within the activated filament, the DREN nuclease domains form tetramers that are poised to cleave dsDNA. We show that other SPARDAs also form filaments during activation. Furthermore, we identify the presence of the beta-relay in pAgo from all clades, providing new insights into the structural mechanisms of pAgo proteins. Taken together, these findings reveal the detailed structural mechanism of SPARDA and highlight the importance of the beta-relay mechanism in signal transduction in Argonautes.
History
DepositionMar 4, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 17, 2025Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Dec 17, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: DUF4365 domain-containing protein
B: Protein argonaute
C: DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
D: RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
E: DUF4365 domain-containing protein
F: Protein argonaute
G: DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
H: RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
I: DUF4365 domain-containing protein
J: Protein argonaute
K: DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
L: RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
M: DUF4365 domain-containing protein
N: Protein argonaute
O: DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
P: RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
Q: DUF4365 domain-containing protein
R: Protein argonaute
1: DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
2: RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
U: DUF4365 domain-containing protein
V: Protein argonaute
3: DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
4: RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
X: Protein argonaute
W: DUF4365 domain-containing protein
5: DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
6: RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
Y: DUF4365 domain-containing protein
Z: Protein argonaute
7: DNA (5'-D(*AP*TP*TP*CP*GP*GP*CP*CP*GP*TP*GP*TP*AP*CP*AP*AP*T)-3')
8: RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')
S: dsDNA substrate, first strand
T: dsDNA substrate, second strand
hetero molecules


Theoretical massNumber of molelcules
Total (without water)959,45446
Polymers958,97334
Non-polymers48112
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 2 types, 16 molecules AEIMQUWYBFJNRVXZ

#1: Protein
DUF4365 domain-containing protein


Mass: 52420.254 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Details: N-terminal purification His-tag
Source: (gene. exp.) Xanthobacter autotrophicus Py2 (bacteria)
Gene: Xaut_0433 / Plasmid: pBAD / Production host: Escherichia coli (E. coli) / References: UniProt: A7ICE8
#2: Protein
Protein argonaute


Mass: 53589.664 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xanthobacter autotrophicus Py2 (bacteria)
Gene: Xaut_0434 / Plasmid: pBAD / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A7ICE9

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DsDNA substrate, ... , 2 types, 2 molecules ST

#5: DNA chain dsDNA substrate, first strand


Mass: 12994.325 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: first strand of dsDNA substrate / Source: (synth.) Escherichia coli (E. coli)
#6: DNA chain dsDNA substrate, second strand


Mass: 12862.349 Da / Num. of mol.: 1 / Source method: obtained synthetically / Details: complementary strand of dsDNA substrate / Source: (synth.) Escherichia coli (E. coli)

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DNA chain / RNA chain / Non-polymers , 3 types, 28 molecules CGKO1357DHLP2468

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


Mass: 5202.384 Da / Num. of mol.: 8 / Source method: obtained synthetically / Details: DNA target strand / Source: (synth.) Escherichia coli (E. coli)
#4: RNA chain
RNA (5'-R(P*AP*UP*UP*GP*UP*AP*CP*AP*CP*GP*GP*CP*CP*GP*AP*AP*U)-3')


Mass: 5427.286 Da / Num. of mol.: 8 / Source method: obtained synthetically / Details: 5'-P RNA guide strand / Source: (synth.) Escherichia coli (E. coli)
#7: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeDetailsEntity IDParent-IDSource
1Filament assembly of activated XauSPARDA binary monomers in the presence of dsDNA substrateCOMPLEXSPARDA binary monomers are activated upon binding of guide 5'p-RNA and complementary DNA target (component 1). Activated monomers oligomerise into filament-like structure, that supports the tetrameric assembly of DREN-domains (component 2) which unspecifically binds dsDNA substrate#1-#60RECOMBINANT
2Activated XauSPARDA binary monomer containing bound 5'p-RNA guide and complementary DNA targetCOMPLEXXauSPARDA binary monomer binds 5'p-RNA guide and complementary DNA target strand in the presence of calcium ion#1-#41RECOMBINANT
3DREN-domains tetramer with bound dsDNA substrateCOMPLEXDREN-domains tetramer assembled upon oligomerization of activated binary monomers in the presence of dsDNA and calcium#1, #5-#61RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
111.028 MDaNO
210.127 MDaNO
310.22 MDaNO
41NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Xanthobacter autotrophicus Py2 (bacteria)78245
32Xanthobacter autotrophicus Py2 (bacteria)78245
43Xanthobacter autotrophicus Py2 (bacteria)78245
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrainPlasmid
21Escherichia coli (E. coli)562BL21pBAD
32Escherichia coli (E. coli)562BL21pBAD
43Escherichia coli (E. coli)562BL21pBAD
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMtris HCl1
2100 mMNaCl1
32 mMCACl21
SpecimenConc.: 1.96 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Solution containing activated XauSPARDA monomers was mixed with solution containing dsDNA substrate and centrifugated before applying on the grid.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal magnification: 92000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: OTHER
Image recordingAverage exposure time: 46.33 sec. / Electron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1946

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARCv.4.2.1particle selection
4cryoSPARCv.4.2.1CTF correction
7UCSF Chimera1.17model fittingFitting of monomrs into map
9cryoSPARCv.4.2.1initial Euler assignment
10cryoSPARCv.4.2.1final Euler assignment
11cryoSPARCv.4.2.1classification
12cryoSPARCv.4.2.13D reconstruction
13PHENIX1.20_4459model refinementphenix.real_space_refine
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 6077663 / Details: Template picking
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204199 / Algorithm: FOURIER SPACE / Details: Local refinement within mask / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 131 / Protocol: RIGID BODY FIT / Space: REAL
Atomic model building

3D fitting-ID: 1 / Accession code: 9QBQ / PDB-ID: 9QBQ

9qbq

/ Source name: PDB / Type: experimental model

IDPdb chain-IDChain-IDDetails
1CCapo XauSPARDA DREN-APAZ subunit
2DDapo XauSPARDA Ago subunit

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