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- EMDB-49407: Cryo-EM structure of SIWI-piRNA-target(25-nt)-GTSF1 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-49407
TitleCryo-EM structure of SIWI-piRNA-target(25-nt)-GTSF1 complex
Map data
Sample
  • Complex: SIWI-piRNA-target(25-nt)-GTSF1 complex
    • Protein or peptide: Piwi-like protein Siwi
    • RNA: piRNA
    • RNA: target RNA (25-nt)
    • Protein or peptide: GTSF1
  • Ligand: MAGNESIUM ION
KeywordsSIWI / GTSF1 / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


female sex determination / PET complex / secondary piRNA processing / piRNA binding / piRNA-mediated gene silencing by mRNA destabilization / piRNA processing / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / RNA endonuclease activity / meiotic cell cycle / spermatogenesis ...female sex determination / PET complex / secondary piRNA processing / piRNA binding / piRNA-mediated gene silencing by mRNA destabilization / piRNA processing / Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters / RNA endonuclease activity / meiotic cell cycle / spermatogenesis / cell differentiation / magnesium ion binding / metal ion binding / cytoplasm
Similarity search - Function
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 / Piwi domain profile. / Piwi domain / Piwi domain / Piwi / PAZ domain superfamily / PAZ ...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 / Piwi domain profile. / Piwi domain / Piwi domain / Piwi / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain / Zinc finger C2H2 superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
CHHC U11-48K-type domain-containing protein / Piwi-like protein Siwi
Similarity search - Component
Biological speciesBombyx mori (domestic silkworm) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsSarkar S / Gebert LFR / MacRae IJ
Funding support United States, 1 items
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 24, 2025-
Header (metadata) releaseSep 17, 2025-
Map releaseSep 17, 2025-
UpdateOct 1, 2025-
Current statusOct 1, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49407.png

Downloads & links

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Map

FileDownload / File: emd_49407.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.91 Å/pix.
x 300 pix.
= 273. Å		0.91 Å/pix.
x 300 pix.
= 273. Å		0.91 Å/pix.
x 300 pix.
= 273. Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.91 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-2.4470274 - 3.4949884
Average (Standard dev.)0.0005439738 (±0.070845895)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 273.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_49407_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_49407_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SIWI-piRNA-target(25-nt)-GTSF1 complex

EntireName: SIWI-piRNA-target(25-nt)-GTSF1 complex
Components
  • Complex: SIWI-piRNA-target(25-nt)-GTSF1 complex
    • Protein or peptide: Piwi-like protein Siwi
    • RNA: piRNA
    • RNA: target RNA (25-nt)
    • Protein or peptide: GTSF1
  • Ligand: MAGNESIUM ION

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Supramolecule #1: SIWI-piRNA-target(25-nt)-GTSF1 complex

SupramoleculeName: SIWI-piRNA-target(25-nt)-GTSF1 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Bombyx mori (domestic silkworm)

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Macromolecule #1: Piwi-like protein Siwi

MacromoleculeName: Piwi-like protein Siwi / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
EC number: Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 101.484242 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSEPRGRGRA RGRAGRGGDG GGPAPRRPGE QAGPSQQSMP PGPRPQPPSG WGPQSSVPPV RAGVPTPTAQ AGRASHRVTP TTHEHPGDI DVQQRMQKLE LGPHSSGGGD ASSVVGRGSR RGGGRVLPET ISILRTRPEA VTSKKGTSGT PLDLLANYFT V ETTPKWGL ...String:
MSEPRGRGRA RGRAGRGGDG GGPAPRRPGE QAGPSQQSMP PGPRPQPPSG WGPQSSVPPV RAGVPTPTAQ AGRASHRVTP TTHEHPGDI DVQQRMQKLE LGPHSSGGGD ASSVVGRGSR RGGGRVLPET ISILRTRPEA VTSKKGTSGT PLDLLANYFT V ETTPKWGL YQYHVDISPE EDSTGVRKAL MRVHSKTLGG YLFDGTVLYT VNRLHPDPME LYSDRKTDNE RMRILIKLTC EV SPGDYHY IQIFNIIIRK CFNLLKLQLM GRDYFDPEAK IDIPEFKLQI WPGYKTTINQ YEDRLLLVTE IAHKVLRMDT VLQ MLSEYA ATKGNNYKKI FLEDVVGKIV MTDYNKRTYR VDDVAWNVSP KSTFKMRDEN ITYIEYYYKK YNLRIQDPGQ PLLI SRSKP REIRAGLPEL IYLVPELCRQ TGLSDEMRAN FKLMRSLDVH TKIGPDKRIE KLNNFNRRFT STPEVVEELA TWSLK LSKE LVKIKGRQLP PENIIQANNV KYPAGDTTEG WTRDMRSKHL LAIAQLNSWV VITPERQRRD TESFIDLIIK TGGGVG FRM RSPDLVVIRH DGPIEYANMC EEVIARKNPA LILCVLARNY ADRYEAIKKK CTVDRAVPTQ VVCARNMSSK SAMSIAT KV AIQINCKLGG SPWTVDIPLP SLMVVGYDVC HDTRSKEKSF GAFVATLDKQ MTQYYSIVNA HTSGEELSSH MGFNIASA V KKFREKNGTY PARIFIYRDG VGDGQIPYVH SHEVAEIKKK LAEIYAGVEI KLAFIIVSKR INTRIFVQRG RSGENPRPG TVIDDVVTLP ERYDFYLVSQ NVREGTIAPT SYNVIEDTTG LNPDRIQRLT YKLTHLYFNC SSQVRVPSVC QYAHKLAFLA ANSLHNQPH YSLNETLYFL

UniProtKB: Piwi-like protein Siwi

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Macromolecule #4: GTSF1

MacromoleculeName: GTSF1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Bombyx mori (domestic silkworm)
Molecular weightTheoretical: 17.358834 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MEVTIANPKP NQMITCPYEK AHIVEHYRMH IHLQKCRKQH PACNKVQCPF DATHVVNDVE LDFHVTVCPK RHMLDTQLYI TDDEYRPTV EVHATPVLPS DENWDDETST SYIPDPSKKG AHIITKAKGL TRSERQSLRK ELIKTYRPLE

UniProtKB: CHHC U11-48K-type domain-containing protein

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Macromolecule #2: piRNA

MacromoleculeName: piRNA / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.111833 KDa
SequenceString:
UGGAGUGUGA CAAUGGUGUU UGAU(A2M)

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Macromolecule #3: target RNA (25-nt)

MacromoleculeName: target RNA (25-nt) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 8.505142 KDa
SequenceString:
UAUCAAACAC CAUUGUCACA CUCCAAA

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.89 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTRIStris(hydroxymethyl)aminomethane
100.0 mMNaClsodium chloride
0.5 mMTCEPtris(2-carboxyethyl)phosphine
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 25 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.026000000000000002 kPa / Details: 15 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number real images: 4687 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 161717
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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Atomic model buiding 1

Initial model
PDB IDChain

5guh

chain_id: A, source_name: PDB, initial_model_type: experimental model
chain_id: E, source_name: AlphaFold, initial_model_type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-9nhb:
Cryo-EM structure of SIWI-piRNA-target(25-nt)-GTSF1 complex

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