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- PDB-9om4: Cryo-EM structure of filament form Acidithiobacillus caldus (Aca)... -

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Basic information

Entry
Database: PDB / ID: 9om4
TitleCryo-EM structure of filament form Acidithiobacillus caldus (Aca) short prokaryotic argonautes, HNH-associated (SPARHA) with gRNA and tDNA
Components
  • DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
  • HNH endonuclease
  • Prokaryotic argonaute MID-PIWI family protein
  • RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
KeywordsIMMUNE SYSTEM/DNA/RNA / SPARHA / prokaryotic argonaute / nuclease / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA-RNA complex
Function / homology: / DNA / DNA (> 10) / RNA / RNA (> 10)
Function and homology information
Biological speciesAcidithiobacillus caldus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsMurakami, K.S. / Narwal, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM156623 United States
CitationJournal: Nat Commun / Year: 2025
Title: Argonaute-HNH filaments triggered by invader DNA confer bacterial immunity.
Authors: Anna Kanevskaya / Manju Narwal / Lidiya Lisitskaya / Andrey V Moiseenko / Olga S Sokolova / Nikolai N Sluchanko / Katsuhiko S Murakami / Andrey Kulbachinskiy /
Abstract: Argonaute proteins provide innate immunity in all domains of life through guide-dependent recognition and cleavage of invader nucleic acids. Many short prokaryotic Argonautes (pAgos) lack nuclease ...Argonaute proteins provide innate immunity in all domains of life through guide-dependent recognition and cleavage of invader nucleic acids. Many short prokaryotic Argonautes (pAgos) lack nuclease activity and are instead co-encoded with tentative nuclease effectors, but their activation mechanisms remain unknown. Here, we characterize SPARHA systems (short prokaryotic argonautes, HNH-associated), containing HNH nuclease effectors. RNA-guided target DNA recognition by pAgo induces formation of SPARHA filaments with a chain of double nuclease sites formed by HNH tetramers, leading to indiscriminate collateral degradation of DNA and protecting bacterial population from invaders. We show that the assembly of filaments proceeds via a universal activation pathway involving a cascade of target-induced conformational changes in SPARHA, conserved in other short pAgo systems containing various types of effectors. pAgos and associated effectors act as modular defense systems that translate recognition of specific DNA into immune response through assembly of supramolecular complexes, deleterious for invaders and potentially useful for biotechnology.
History
DepositionMay 13, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 22, 2025Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Oct 22, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
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Revision 1.1May 13, 2026Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update
Revision 1.1May 13, 2026Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Database references / Experimental summary / Data content type: EM metadata / EM metadata / EM metadata / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: HNH endonuclease
B: Prokaryotic argonaute MID-PIWI family protein
C: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
D: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
E: HNH endonuclease
F: Prokaryotic argonaute MID-PIWI family protein
G: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
H: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
I: HNH endonuclease
J: Prokaryotic argonaute MID-PIWI family protein
K: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
L: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
M: HNH endonuclease
N: Prokaryotic argonaute MID-PIWI family protein
O: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
P: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)458,70720
Polymers458,48816
Non-polymers2204
Water00
1
A: HNH endonuclease
B: Prokaryotic argonaute MID-PIWI family protein
C: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
D: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,6775
Polymers114,6224
Non-polymers551
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, C2 symmetry related to chain IDs M,N,O,P
TypeNameSymmetry operationNumber
identity operation1_5551
3
E: HNH endonuclease
F: Prokaryotic argonaute MID-PIWI family protein
G: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
H: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,6775
Polymers114,6224
Non-polymers551
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
4
I: HNH endonuclease
J: Prokaryotic argonaute MID-PIWI family protein
K: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
L: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,6775
Polymers114,6224
Non-polymers551
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
5
M: HNH endonuclease
N: Prokaryotic argonaute MID-PIWI family protein
O: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
P: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)114,6775
Polymers114,6224
Non-polymers551
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
HNH endonuclease


Mass: 50263.348 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Acidithiobacillus caldus short prokaryotic Argonaute HNH-APAZ endonuclease
Source: (gene. exp.) Acidithiobacillus caldus (bacteria) / Plasmid: pBAD_AcaAgo / Production host: Escherichia coli BL21(DE3) (bacteria)
#2: Protein
Prokaryotic argonaute MID-PIWI family protein


Mass: 53150.621 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: Acidithiobacillus caldus argonaute MID-PIWI family protein
Source: (gene. exp.) Acidithiobacillus caldus (bacteria) / Plasmid: pBAD_AcaAgo / Production host: Escherichia coli BL21(DE3) (bacteria)
#3: RNA chain
RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')


Mass: 5720.355 Da / Num. of mol.: 4 / Source method: obtained synthetically / Details: guide RNA with 5'-phosphate group / Source: (synth.) Acidithiobacillus caldus (bacteria)
#4: DNA chain
DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')


Mass: 5487.598 Da / Num. of mol.: 4 / Source method: obtained synthetically / Details: target DNA / Source: (synth.) Acidithiobacillus caldus (bacteria)
#5: Chemical
ChemComp-MN / MANGANESE (II) ION


Mass: 54.938 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mn / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

Component
IDNameTypeDetails (eV)Entity IDParent-IDSource
1Acidithiobacillus caldus SPARHA complex with guide RNA and target DNACOMPLEXTetramer with C2 symmetry#1-#40MULTIPLE SOURCES
2SPARHA complexCOMPLEX#1-#21RECOMBINANT
3guide RNA and target DNACOMPLEX#3-#41SYNTHETIC
Molecular weightValue: 103.14 kDa/nm / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Acidithiobacillus caldus (bacteria)33059
33Acidithiobacillus caldus (bacteria)33059
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(DE3)
Buffer solutionpH: 7 / Details: 20 mM Tris-HCl, 50 mM NaCl, and MnCl2 (0.1 mM)
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails (eV)
4cryoSPARC4.6.2CTF correctionPatch CTF Estimation
7UCSF ChimeraX1.9model fitting
12cryoSPARC4.6.23D reconstructionLocal refinement
13cryoSPARC4.6.23D reconstructionNon-uniform refinement
14PHENIXdev_5330model refinementminimization_global, local_grid_search, secondary structure restraints
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 103628 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 57.6 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.004227944
ELECTRON MICROSCOPYf_angle_d0.845438468
ELECTRON MICROSCOPYf_chiral_restr0.05264204
ELECTRON MICROSCOPYf_plane_restr0.00784452
ELECTRON MICROSCOPYf_dihedral_angle_d17.86465308

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