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- PDB-9qbp: Crystal structure of Enhydrobacter aerosaccus apo SPARDA complex -

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Basic information

Entry
Database: PDB / ID: 9qbp
TitleCrystal structure of Enhydrobacter aerosaccus apo SPARDA complex
Components
  • DUF4365 domain-containing protein
  • Protein argonaute
KeywordsRNA BINDING PROTEIN / Protein-nucleic acid interactions / Argonaute / pAgo / guide and target specificity
Function / homology
Function and homology information


nucleic acid binding
Similarity search - Function
Domain of unknown function DUF4365 / Domain of unknown function (DUF4365) / Piwi domain profile. / Piwi domain / Piwi / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
BETA-MERCAPTOETHANOL / DUF4365 domain-containing protein / Protein argonaute
Similarity search - Component
Biological speciesEnhydrobacter aerosaccus (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 2.45 Å
AuthorsManakova, E.N. / Grazulis, S. / Zaremba, M.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
iNEXTH2020 Grant # 653706European Union
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 3, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 17, 2025Provider: repository / Type: Initial release
Revision 1.1Dec 24, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DUF4365 domain-containing protein
B: Protein argonaute
C: DUF4365 domain-containing protein
D: Protein argonaute
hetero molecules


Theoretical massNumber of molelcules
Total (without water)215,24115
Polymers214,6374
Non-polymers60511
Water6,954386
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)108.179, 127.241, 191.336
Angle α, β, γ (deg.)90.00, 90.00, 90.00
Int Tables number19
Space group name H-MP212121

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Components

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Protein , 2 types, 4 molecules ACBD

#1: Protein DUF4365 domain-containing protein


Mass: 53186.844 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enhydrobacter aerosaccus (bacteria) / Strain: ATCC 27094 / Gene: SAMN02745126_06076 / Plasmid: pBAD_HisA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1T4TDU6
#2: Protein Protein argonaute


Mass: 54131.445 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enhydrobacter aerosaccus (bacteria) / Strain: ATCC 27094 / Gene: SAMN02745126_06077 / Plasmid: pBAD_HisA / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1T4TEP4

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Non-polymers , 4 types, 397 molecules

#3: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-TRS / 2-AMINO-2-HYDROXYMETHYL-PROPANE-1,3-DIOL / TRIS BUFFER


Mass: 122.143 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H12NO3 / Comment: pH buffer*YM
#5: Chemical ChemComp-BME / BETA-MERCAPTOETHANOL


Mass: 78.133 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C2H6OS
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 386 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 3.07 Å3/Da / Density % sol: 59.91 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: Crystallization solution: TrisHCl pH 8.5 0.1M; PEG400 20% (w/v) Cryoprotection solution: TrisHCl pH 8 0.1M; PEG400 35%; Ammonium acetate 0.2M
PH range: 8-8.5

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: PETRA III, EMBL c/o DESY / Beamline: P13 (MX1) / Wavelength: 0.98 Å
DetectorType: DECTRIS PILATUS 12M / Detector: PIXEL / Date: Jul 1, 2019
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.98 Å / Relative weight: 1
ReflectionResolution: 2.45→127.24 Å / Num. obs: 195126 / % possible obs: 99.9 % / Redundancy: 6.6 % / Biso Wilson estimate: 61.462 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.08 / Rpim(I) all: 0.034 / Rrim(I) all: 0.088 / Χ2: 1.01 / Net I/av σ(I): 6.8 / Net I/σ(I): 12.9
Reflection shellResolution: 2.45→2.49 Å / Redundancy: 6.7 % / Rmerge(I) obs: 0.826 / Mean I/σ(I) obs: 2.2 / Num. unique obs: 4792 / CC1/2: 0.881 / Rpim(I) all: 0.343 / Rrim(I) all: 0.895 / Χ2: 1.04 / % possible all: 100

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Processing

Software
NameVersionClassification
PHENIX(1.20_4459: ???)refinement
XDSVERSION Mar 15, 2019 BUILT=20190315data reduction
Aimlessversion 0.7.4 : 13/12/18data scaling
MOLREPversion 11.6.04phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT / Resolution: 2.45→94.17 Å / SU ML: 0.34 / Cross valid method: FREE R-VALUE / σ(F): 1.18 / Phase error: 29.36
RfactorNum. reflection% reflectionSelection details
Rfree0.2479 18820 10.06 %random selection
Rwork0.2049 ---
obs0.2092 187049 99.74 %-
Solvent computationIon probe radii: 1.1 Å / Shrinkage radii: 0.9 Å / VDW probe radii: 1.1 Å
Displacement parametersBiso mean: 65.6 Å2
Refinement stepCycle: LAST / Resolution: 2.45→94.17 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms13061 0 0 386 13447
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
2.45-2.480.36476300.32195511X-RAY DIFFRACTION100
2.48-2.510.36066300.31135631X-RAY DIFFRACTION100
2.51-2.540.3576060.3025615X-RAY DIFFRACTION99
2.54-2.570.3566380.28915598X-RAY DIFFRACTION100
2.57-2.60.33196260.28755620X-RAY DIFFRACTION100
2.6-2.640.33576270.27445625X-RAY DIFFRACTION100
2.64-2.680.31886350.27565608X-RAY DIFFRACTION100
2.68-2.720.3546120.28425580X-RAY DIFFRACTION100
2.72-2.760.32836380.27995649X-RAY DIFFRACTION100
2.76-2.80.33075670.27165631X-RAY DIFFRACTION100
2.8-2.850.35416460.25915668X-RAY DIFFRACTION100
2.85-2.90.30476320.24855567X-RAY DIFFRACTION100
2.9-2.960.30565830.245651X-RAY DIFFRACTION100
2.96-3.020.28866720.23235563X-RAY DIFFRACTION100
3.02-3.090.27136600.23285583X-RAY DIFFRACTION99
3.09-3.160.29295530.24435651X-RAY DIFFRACTION100
3.16-3.240.32156460.2575598X-RAY DIFFRACTION100
3.24-3.330.26656560.2295595X-RAY DIFFRACTION100
3.33-3.420.25366480.21895635X-RAY DIFFRACTION100
3.42-3.530.26525960.21085654X-RAY DIFFRACTION100
3.53-3.660.2796110.19955581X-RAY DIFFRACTION100
3.66-3.810.236280.19725628X-RAY DIFFRACTION100
3.81-3.980.22866250.19055653X-RAY DIFFRACTION100
3.98-4.190.21466210.17995554X-RAY DIFFRACTION99
4.19-4.450.196000.16275634X-RAY DIFFRACTION100
4.45-4.80.21076550.15815601X-RAY DIFFRACTION100
4.8-5.280.18376470.15375578X-RAY DIFFRACTION100
5.28-6.040.21736680.18025591X-RAY DIFFRACTION100
6.04-7.610.24046450.1835601X-RAY DIFFRACTION100
7.61-94.170.19796190.18065575X-RAY DIFFRACTION99

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