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- PDB-9qbl: Crystal structure of Xanthobacter autotrophicus SPARDA mutant lac... -

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Basic information

Entry
Database: PDB / ID: 9qbl
TitleCrystal structure of Xanthobacter autotrophicus SPARDA mutant lacking DREN nuclease domains
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 / Piwi / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
ACETIC ACID / BETA-MERCAPTOETHANOL / DI(HYDROXYETHYL)ETHER / D(-)-TARTARIC ACID / DUF4365 domain-containing protein / Protein argonaute
Similarity search - Component
Biological speciesXanthobacter autotrophicus Py2 (bacteria)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MAD / Resolution: 1.5 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

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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)184,47332
Polymers182,0784
Non-polymers2,39528
Water22,1221228
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, SAXS, electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Unit cell
Length a, b, c (Å)72.706, 155.396, 161.338
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: 37138.730 Da / Num. of mol.: 2
Mutation: Deletion of N-terminal DUF4365 domain (1-153) and N-terminal His-tag
Source method: isolated from a genetically manipulated source
Details: Deletion of N-terminal DUF4365 domain (1-153) and N-terminal purification bHis-tag
Source: (gene. exp.) Xanthobacter autotrophicus Py2 (bacteria)
Gene: Xaut_0433 / Plasmid: pLATE / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A7ICE8
#2: Protein Protein argonaute


Mass: 53900.258 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Xanthobacter autotrophicus Py2 (bacteria)
Gene: Xaut_0434 / Plasmid: plasmid / Details (production host): pLATE / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A7ICE9

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Non-polymers , 8 types, 1256 molecules

#3: Chemical
ChemComp-ACY / ACETIC ACID


Mass: 60.052 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C2H4O2
#4: Chemical
ChemComp-GOL / GLYCEROL / GLYCERIN / PROPANE-1,2,3-TRIOL


Mass: 92.094 Da / Num. of mol.: 7 / Source method: obtained synthetically / Formula: C3H8O3
#5: 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
#6: Chemical
ChemComp-PEG / DI(HYDROXYETHYL)ETHER


Mass: 106.120 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C4H10O3
#7: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Cl
#8: Chemical
ChemComp-BME / BETA-MERCAPTOETHANOL


Mass: 78.133 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C2H6OS
#9: Chemical ChemComp-TAR / D(-)-TARTARIC ACID


Mass: 150.087 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C4H6O6
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 1228 / Source method: isolated from a natural source / Formula: H2O

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Details

Has ligand of interestN
Has protein modificationY

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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: 2.52 Å3/Da / Density % sol: 51.25 %
Crystal growTemperature: 280 K / Method: vapor diffusion, sitting drop / pH: 8.5
Details: Protein concentration 6.4 mg/ml. Reservoir: Tris-Bicine pH 8.5 0.1 M; Ammonium Tartrate 0.25M; PEG5000 mme 9% (w/v); Glycerol 2% (v/v). Seeding with microcrystals
Temp details: cold room

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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: 0.97970,0.97980,0.98000
DetectorType: DECTRIS PILATUS 12M / Detector: PIXEL / Date: Jul 1, 2019
RadiationProtocol: MAD / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelength
IDWavelength (Å)Relative weight
10.97971
20.97981
30.981
ReflectionResolution: 1.5→161.34 Å / Num. obs: 284306 / % possible obs: 98.2 % / Redundancy: 16.5 % / Biso Wilson estimate: 28.703 Å2 / CC1/2: 0.998 / Rmerge(I) obs: 0.12 / Rpim(I) all: 0.043 / Rrim(I) all: 0.127 / Net I/av σ(I): 2.8 / Net I/σ(I): 10.3
Reflection shellResolution: 1.5→1.53 Å / Redundancy: 4 % / Rmerge(I) obs: 1.512 / Mean I/σ(I) obs: 0.6 / Num. unique obs: 8990 / CC1/2: 0.023 / Rpim(I) all: 0.956 / Rrim(I) all: 1.86 / % possible all: 63.3

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Processing

Software
NameVersionClassification
REFMAC5.8.0258refinement
XDSdata reduction
Aimlessversion 7.0.020data scaling
Auto-Rickshawphasing
RefinementMethod to determine structure: MAD / Resolution: 1.5→80.67 Å / Cor.coef. Fo:Fc: 0.973 / Cor.coef. Fo:Fc free: 0.959 / SU B: 3.007 / SU ML: 0.095 / Cross valid method: THROUGHOUT / ESU R: 0.084 / ESU R Free: 0.09 / Stereochemistry target values: MAXIMUM LIKELIHOOD / Details: HYDROGENS HAVE BEEN USED IF PRESENT IN THE INPUT
RfactorNum. reflection% reflectionSelection details
Rfree0.237 27155 10.2 %RANDOM
Rwork0.196 ---
obs0.2 240032 92.4 %-
Solvent computationIon probe radii: 0.8 Å / Shrinkage radii: 0.8 Å / VDW probe radii: 1.4 Å / Solvent model: MASK
Displacement parametersBiso mean: 41.48 Å2
Baniso -1Baniso -2Baniso -3
1-0.74 Å20 Å20 Å2
2---0.11 Å20 Å2
3----0.63 Å2
Refinement stepCycle: LAST / Resolution: 1.5→80.67 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms11648 0 146 1228 13022
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
X-RAY DIFFRACTIONr_bond_refined_d0.0080.01212447
X-RAY DIFFRACTIONr_bond_other_d
X-RAY DIFFRACTIONr_angle_refined_deg1.5451.64416901
X-RAY DIFFRACTIONr_angle_other_deg
X-RAY DIFFRACTIONr_dihedral_angle_1_deg6.36251561
X-RAY DIFFRACTIONr_dihedral_angle_2_deg27.43720.192728
X-RAY DIFFRACTIONr_dihedral_angle_3_deg13.874151998
X-RAY DIFFRACTIONr_dihedral_angle_4_deg19.18915137
X-RAY DIFFRACTIONr_chiral_restr0.1090.21566
X-RAY DIFFRACTIONr_gen_planes_refined0.0090.029808
X-RAY DIFFRACTIONr_gen_planes_other
X-RAY DIFFRACTIONr_nbd_refined
X-RAY DIFFRACTIONr_nbd_other
X-RAY DIFFRACTIONr_nbtor_refined
X-RAY DIFFRACTIONr_nbtor_other
X-RAY DIFFRACTIONr_xyhbond_nbd_refined
X-RAY DIFFRACTIONr_xyhbond_nbd_other
X-RAY DIFFRACTIONr_metal_ion_refined
X-RAY DIFFRACTIONr_metal_ion_other
X-RAY DIFFRACTIONr_symmetry_vdw_refined
X-RAY DIFFRACTIONr_symmetry_vdw_other
X-RAY DIFFRACTIONr_symmetry_hbond_refined
X-RAY DIFFRACTIONr_symmetry_hbond_other
X-RAY DIFFRACTIONr_symmetry_metal_ion_refined
X-RAY DIFFRACTIONr_symmetry_metal_ion_other
X-RAY DIFFRACTIONr_mcbond_it3.5053.8416112
X-RAY DIFFRACTIONr_mcbond_other
X-RAY DIFFRACTIONr_mcangle_it4.7785.727701
X-RAY DIFFRACTIONr_mcangle_other
X-RAY DIFFRACTIONr_scbond_it4.9654.2876335
X-RAY DIFFRACTIONr_scbond_other
X-RAY DIFFRACTIONr_scangle_it
X-RAY DIFFRACTIONr_scangle_other
X-RAY DIFFRACTIONr_long_range_B_refined8.7552.87618821
X-RAY DIFFRACTIONr_long_range_B_other
X-RAY DIFFRACTIONr_rigid_bond_restr
X-RAY DIFFRACTIONr_sphericity_free
X-RAY DIFFRACTIONr_sphericity_bonded
LS refinement shellResolution: 1.5→1.54 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0.609 1538 -
Rwork0.606 13131 -
obs--69.07 %

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