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- PDB-9nhs: Cryo-EM structure of SIWI-piRNA-target(39-nt)-GTSF1-Maelstrom-Spi... -

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Basic information

Entry
Database: PDB / ID: 9nhs
TitleCryo-EM structure of SIWI-piRNA-target(39-nt)-GTSF1-Maelstrom-Spindle-E complex
Components
  • GTSF1
  • Maelstrom
  • Piwi-like protein Siwi
  • Spindle-E
  • piRNA
  • target RNA (39-nt)
KeywordsRNA BINDING PROTEIN/RNA / SIWI / GTSF1 / Maelstrom / RNA BINDING PROTEIN / Spindle-E / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


female sex determination / regulation of miRNA-mediated gene silencing / PET complex / secondary piRNA processing / piRNA binding / piRNA-mediated gene silencing by mRNA destabilization / piRNA processing / male meiotic nuclear division / regulatory ncRNA-mediated gene silencing / P granule ...female sex determination / regulation of miRNA-mediated gene silencing / PET complex / secondary piRNA processing / piRNA binding / piRNA-mediated gene silencing by mRNA destabilization / piRNA processing / male meiotic nuclear division / regulatory ncRNA-mediated gene silencing / P granule / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / RNA endonuclease activity / meiotic cell cycle / helicase activity / spermatogenesis / sequence-specific DNA binding / cell differentiation / RNA helicase / negative regulation of DNA-templated transcription / magnesium ion binding / RNA binding / ATP binding / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Maelstrom domain / Protein maelstrom / piRNA pathway germ-plasm component / TRM13/UPF0224 family, U11-48K-like CHHC zinc finger domain / U11-48K-like CHHC zinc finger / Zinc finger CHHC U11-48K-type profile. / Piwi, N-terminal domain / Tudor domain / HMG-box domain / Piwi domain profile. ...Maelstrom domain / Protein maelstrom / piRNA pathway germ-plasm component / TRM13/UPF0224 family, U11-48K-like CHHC zinc finger domain / U11-48K-like CHHC zinc finger / Zinc finger CHHC U11-48K-type profile. / Piwi, N-terminal domain / Tudor domain / HMG-box domain / Piwi domain profile. / Piwi domain / Piwi domain / Piwi / PAZ domain superfamily / PAZ / PAZ domain / Helicase associated domain (HA2), ratchet-like / Tudor domain / Helicase-associated domain / Helicase associated domain (HA2) Add an annotation / PAZ domain profile. / PAZ domain / SNase-like, OB-fold superfamily / HMG boxes A and B DNA-binding domains profile. / High mobility group box domain / High mobility group box domain superfamily / Zinc finger C2H2 superfamily / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
RNA / RNA (> 10) / CHHC U11-48K-type domain-containing protein / Probable ATP-dependent RNA helicase spindle-E / HMG box domain-containing protein / Piwi-like protein Siwi
Similarity search - Component
Biological speciesBombyx mori (domestic silkworm)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsSarkar, S. / Gebert, L.F.R. / MacRae, I.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM127090 United States
CitationJournal: Mol Cell / Year: 2025
Title: A conserved PIWI silencing complex detects piRNA-target engagement.
Authors: Dipayan De / Sucharita Sarkar / Luca F R Gebert / Timothy Wiryaman / Todd A Anzelon / Ian J MacRae /
Abstract: In animal germ cells, PIWI proteins use piRNAs to detect active selfish genetic elements. Base-pairing to a piRNA defines transposon recognition, but how this interaction triggers a defensive ...In animal germ cells, PIWI proteins use piRNAs to detect active selfish genetic elements. Base-pairing to a piRNA defines transposon recognition, but how this interaction triggers a defensive response remains unclear. Here, we identify a transposon recognition complex composed of the silkworm proteins Siwi, GTSF1, and Maelstrom. Biochemical and cryo-electron microscopy (cryo-EM) analyses show that extended piRNA-target pairing locks Siwi in a conformation that recruits GTSF1 and Maelstrom. Extended piRNA-target pairing is recognized by the N-terminal helix of Maelstrom and the first zinc finger of GTSF1, which act together to hold Siwi in an endonucleolytically active state. The resulting activated complex, termed Siwi, rapidly cleaves target RNAs and recruits the piRNA biogenesis factor Spindle-E. Structural predictions reveal related complexes in animals ranging from humans to sponges, indicating PIWI assembly is a conserved transposon recognition mechanism employed broadly across the metazoan kingdom.
History
DepositionFeb 25, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 17, 2025Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Sep 17, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Oct 1, 2025Group: Data collection / Database references / 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: Piwi-like protein Siwi
B: piRNA
C: target RNA (39-nt)
E: GTSF1
F: Maelstrom
G: Spindle-E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)352,80710
Polymers352,5876
Non-polymers2214
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 4 types, 4 molecules AEFG

#1: Protein Piwi-like protein Siwi


Mass: 101484.242 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bombyx mori (domestic silkworm) / Gene: Siwi / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: A8D8P8, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
#4: Protein GTSF1


Mass: 17358.834 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bombyx mori (domestic silkworm) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8R1WGS4
#5: Protein Maelstrom


Mass: 49900.469 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bombyx mori (domestic silkworm) / Production host: Escherichia coli (E. coli) / References: UniProt: A0A8R2M2N1
#6: Protein Spindle-E


Mass: 163275.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bombyx mori (domestic silkworm) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A8R2C579, RNA helicase

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RNA chain , 2 types, 2 molecules BC

#2: RNA chain piRNA


Mass: 8111.833 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: RNA chain target RNA (39-nt)


Mass: 12455.611 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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Non-polymers , 2 types, 4 molecules

#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#8: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn

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Details

Has ligand of interestN
Has protein modificationN

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Experimental details

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Experiment

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

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Sample preparation

ComponentName: SIWI-piRNA-target(39-nt)-GTSF1-Maelstrom-Spindle-E complex
Type: COMPLEX / Entity ID: #1-#6 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Bombyx mori (domestic silkworm)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtris(hydroxymethyl)aminomethaneTRIS1
2100 mMsodium chlorideNaCl1
30.5 mMtris(2-carboxyethyl)phosphineTCEP1
SpecimenConc.: 1.11 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 15 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3412

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Processing

EM software
IDNameVersionCategory
7UCSF ChimeraX1.8model fitting
13PHENIX1.20.1model refinement
14Coot0.9.8.1model refinement
15UCSF ChimeraX1.8model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 83871 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDInitial refinement model-IDSource nameType
15GUH

5guh

A5GUHA1PDBexperimental model
2EAlphaFoldin silico model
3FAlphaFoldin silico model
4GAlphaFoldin silico model

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