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- EMDB-70606: Cryo-EM structure of filament form Acidithiobacillus caldus (Aca)... -

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Basic information

Entry
Database: EMDB / ID: EMD-70606
TitleCryo-EM structure of filament form Acidithiobacillus caldus (Aca) short prokaryotic argonautes, HNH-associated (SPARHA) with gRNA and tDNA
Map dataLocal refinement (C2 symmetry) map using AcaSPARHA tetramer mask
Sample
  • Complex: Acidithiobacillus caldus SPARHA complex with guide RNA and target DNA
    • Complex: SPARHA complex
      • Protein or peptide: HNH endonuclease
      • Protein or peptide: Prokaryotic argonaute MID-PIWI family protein
    • Complex: guide RNA and target DNA
      • RNA: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
      • DNA: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
  • Ligand: MANGANESE (II) ION
KeywordsSPARHA / prokaryotic argonaute / nuclease / IMMUNE SYSTEM / IMMUNE SYSTEM-DNA-RNA complex
Biological speciesAcidithiobacillus caldus (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsMurakami KS / Narwal M
Funding support United States, 1 items
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, 2025-
Header (metadata) releaseOct 22, 2025-
Map releaseOct 22, 2025-
UpdateMay 13, 2026-
Current statusMay 13, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_70606.png

Downloads & links

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Map

FileDownload / File: emd_70606.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal refinement (C2 symmetry) map using AcaSPARHA tetramer mask
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.94 Å/pix.
x 400 pix.
= 377.6 Å		0.94 Å/pix.
x 400 pix.
= 377.6 Å		0.94 Å/pix.
x 400 pix.
= 377.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.944 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.668136 - 0.98603755
Average (Standard dev.)0.00040727193 (±0.025716849)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 377.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_70606_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap-A

Fileemd_70606_half_map_1.map
Annotationhalfmap-A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap-B

Fileemd_70606_half_map_2.map
Annotationhalfmap-B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Acidithiobacillus caldus SPARHA complex with guide RNA and target DNA

EntireName: Acidithiobacillus caldus SPARHA complex with guide RNA and target DNA
Components
  • Complex: Acidithiobacillus caldus SPARHA complex with guide RNA and target DNA
    • Complex: SPARHA complex
      • Protein or peptide: HNH endonuclease
      • Protein or peptide: Prokaryotic argonaute MID-PIWI family protein
    • Complex: guide RNA and target DNA
      • RNA: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
      • DNA: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
  • Ligand: MANGANESE (II) ION

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Supramolecule #1: Acidithiobacillus caldus SPARHA complex with guide RNA and target DNA

SupramoleculeName: Acidithiobacillus caldus SPARHA complex with guide RNA and target DNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: Tetramer with C2 symmetry
Molecular weightTheoretical: 103.14 kDa/nm

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Supramolecule #2: SPARHA complex

SupramoleculeName: SPARHA complex / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Acidithiobacillus caldus (bacteria)

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Supramolecule #3: guide RNA and target DNA

SupramoleculeName: guide RNA and target DNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Acidithiobacillus caldus (bacteria) / Synthetically produced: Yes

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Macromolecule #1: HNH endonuclease

MacromoleculeName: HNH endonuclease / type: protein_or_peptide / ID: 1
Details: Acidithiobacillus caldus short prokaryotic Argonaute HNH-APAZ endonuclease
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Acidithiobacillus caldus (bacteria)
Molecular weightTheoretical: 50.263348 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MAIEPRTKQI LWGRAGATCS FPGCGRNLVR DATPEDREVL VGEIAHIVGQ SKGGPRAEMS VPGGNVDGYD NLILLCHEHH ELVDQQPNT YPVEKLRQYK TDHEEWVRAR LSREQEFEGL TRPEAMVTET VYSNVLPITQ LPHYVYSGPC TVPEAEVKTL I KWPSDKRI ...String:
MAIEPRTKQI LWGRAGATCS FPGCGRNLVR DATPEDREVL VGEIAHIVGQ SKGGPRAEMS VPGGNVDGYD NLILLCHEHH ELVDQQPNT YPVEKLRQYK TDHEEWVRAR LSREQEFEGL TRPEAMVTET VYSNVLPITQ LPHYVYSGPC TVPEAEVKTL I KWPSDKRI LTPYIIRGGR LYAFNDLKDY ESPFATIVDP YSAQRHTLNE WLDKPDEMRW YVELLNRTVN KITGHLRLKL DK EHHRYFF EPDEPGKDKS VTYQSVGGVR SERKVAWNPH FKHNDMPKRY WEHLAVGLRF HRLGDMAWGF AIRPERRFTK DGF ESLEGK ATGKKSTKKK SRMYNFDVLK EVQFWRDFLS QGNPRITCLF GKQALVIDNS LMSAAITWPE ISGDQANRMS ASYE DDLFS LADLQEVSEF DEFDSDTDDL ETEDEVQGED

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Macromolecule #2: Prokaryotic argonaute MID-PIWI family protein

MacromoleculeName: Prokaryotic argonaute MID-PIWI family protein / type: protein_or_peptide / ID: 2
Details: Acidithiobacillus caldus argonaute MID-PIWI family protein
Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Acidithiobacillus caldus (bacteria)
Molecular weightTheoretical: 53.150621 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MKTNTATVRC ELFEEPSLEF ASSRLHLCPK RGITIFGPRS LDMGKRHPDI VRVGFIGTGE TIDSATRWLE SCADGVEGDV ANYRFPGFR ADRGFFSDLS FDAETVERLT LRELEALARP RTKRERFEQA VALLDDKLRL LSQKDRPPDY VVLALPTELV K AQGRVDYY ...String:
MKTNTATVRC ELFEEPSLEF ASSRLHLCPK RGITIFGPRS LDMGKRHPDI VRVGFIGTGE TIDSATRWLE SCADGVEGDV ANYRFPGFR ADRGFFSDLS FDAETVERLT LRELEALARP RTKRERFEQA VALLDDKLRL LSQKDRPPDY VVLALPTELV K AQGRVDYY DKQDGEVHRD LRRAIKASAM KYRLPTQILL QRTTEATPGS KDVDHLSKCA WNFFTGLYYK TGGVPWIPAG LS NGTCYVG ISFHQLLGSK NSGYFTGLAQ AFDEQGNGLV LRGQDFVWDV GKHGNSAHMP APIAEELVSR VLKRYRDELK QSP RRVVIH KTTEFWPEER AGLQSALSGV DQFDLVAVRP TEDIRLFRDA RYPILRGSHI SLGDMHLLYT TGYISALQAF PHGH VPLPL KLTDHLGDSS ISTVLRELMV LTKMNWNSAN FGGLLPITLR FSKLVGEIMK EIPNSVDPLP QFKYYM

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Macromolecule #3: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')

MacromoleculeName: RNA (5'-R(P*AP*UP*UP*UP*GP*GP*UP*UP*GP*CP*GP*GP*UP*GP*CP*UP*CP*U)-3')
type: rna / ID: 3 / Details: guide RNA with 5'-phosphate group / Number of copies: 4
Source (natural)Organism: Acidithiobacillus caldus (bacteria)
Molecular weightTheoretical: 5.720355 KDa
SequenceString:
AUUUGGUUGC GGUGCUCU

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Macromolecule #4: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')

MacromoleculeName: DNA (5'-D(*AP*GP*AP*GP*CP*AP*CP*CP*GP*CP*AP*AP*CP*CP*AP*AP*AP*T)-3')
type: dna / ID: 4 / Details: target DNA / Number of copies: 4 / Classification: DNA
Source (natural)Organism: Acidithiobacillus caldus (bacteria)
Molecular weightTheoretical: 5.487598 KDa
SequenceString:
(DA)(DG)(DA)(DG)(DC)(DA)(DC)(DC)(DG)(DC) (DA)(DA)(DC)(DC)(DA)(DA)(DA)(DT)

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Macromolecule #5: MANGANESE (II) ION

MacromoleculeName: MANGANESE (II) ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MN
Molecular weightTheoretical: 54.938 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration2 mg/mL
BufferpH: 7 / Details: 20 mM Tris-HCl, 50 mM NaCl, and MnCl2 (0.1 mM)
GridModel: Quantifoil R1.2/1.3 / Material: GOLD
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: cryoSPARC (ver. 4.6.2) / Software - details: Patch CTF Estimation / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: AlphaFold predicted model
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF
Software:
Namedetails
cryoSPARC (ver. 4.6.2)Local refinement
cryoSPARC (ver. 4.6.2)Non-uniform refinement

Number images used: 103628
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-9om4:
Cryo-EM structure of filament form Acidithiobacillus caldus (Aca) short prokaryotic argonautes, HNH-associated (SPARHA) with gRNA and tDNA

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