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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: To Be Published
Title: Target-induced Argonaute-HNH filaments confer bacterial immunity
Authors: Murakami, K.S. / Narwal, M. / Kanevskaya, A. / Kulbachinskiy, A.
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
Revision 1.0Oct 22, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Oct 22, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
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
Revision 1.0Oct 22, 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

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
IDNameTypeDetailsEntity 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
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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