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- EMDB-33797: Cryo-EM structure of the EfPiwi (N959K)-piRNA-target ternary complex -

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Basic information

Entry
Database: EMDB / ID: EMD-33797
TitleCryo-EM structure of the EfPiwi (N959K)-piRNA-target ternary complex
Map dataoverall map
Sample
  • Complex: EfPiwi(N959K)-piRNA-16nt target
    • Complex: EfPiwi(N959K)
      • Protein or peptide: Piwi
    • Complex: RNA
      • RNA: piRNA
      • RNA: RNA (5'-R(*UP*CP*CP*AP*UP*GP*UP*UP*GP*AP*UP*GP*GP*UP*AP*A)-3')
  • Ligand: MAGNESIUM ION
KeywordspiRNA / Piwi protein / Argonaute / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


Piwi domain / Piwi domain profile. / Piwi domain / Piwi / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain / PAZ domain profile. / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Biological speciesEphydatia fluviatilis (invertebrata) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsLi ZQ / Liu HB / Wu JP / Shen EZ
Funding support China, 1 items
OrganizationGrant numberCountry
Other privateWestlake Education Foundation (101486021901) China
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Mammalian PIWI-piRNA-target complexes reveal features for broad and efficient target silencing.
Authors: Zhiqing Li / Zhenzhen Li / Yuqi Zhang / Lunni Zhou / Qikui Xu / Lili Li / Lin Zeng / Junchao Xue / Huilin Niu / Jing Zhong / Qilu Yu / Dengfeng Li / Miao Gui / Yongping Huang / Shikui Tu / ...Authors: Zhiqing Li / Zhenzhen Li / Yuqi Zhang / Lunni Zhou / Qikui Xu / Lili Li / Lin Zeng / Junchao Xue / Huilin Niu / Jing Zhong / Qilu Yu / Dengfeng Li / Miao Gui / Yongping Huang / Shikui Tu / Zhao Zhang / Chun-Qing Song / Jianping Wu / En-Zhi Shen /
Abstract: The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure ...The PIWI-interacting RNA (piRNA) pathway is an adaptive defense system wherein piRNAs guide PIWI family Argonaute proteins to recognize and silence ever-evolving selfish genetic elements and ensure genome integrity. Driven by this intensive host-pathogen arms race, the piRNA pathway and its targeted transposons have coevolved rapidly in a species-specific manner, but how the piRNA pathway adapts specifically to target silencing in mammals remains elusive. Here, we show that mouse MILI and human HILI piRNA-induced silencing complexes (piRISCs) bind and cleave targets more efficiently than their invertebrate counterparts from the sponge Ephydatia fluviatilis. The inherent functional differences comport with structural features identified by cryo-EM studies of piRISCs. In the absence of target, MILI and HILI piRISCs adopt a wider nucleic-acid-binding channel and display an extended prearranged piRNA seed as compared with EfPiwi piRISC, consistent with their ability to capture targets more efficiently than EfPiwi piRISC. In the presence of target, the seed gate-which enforces seed-target fidelity in microRNA RISC-adopts a relaxed state in mammalian piRISC, revealing how MILI and HILI tolerate seed-target mismatches to broaden the target spectrum. A vertebrate-specific lysine distorts the piRNA seed, shifting the trajectory of the piRNA-target duplex out of the central cleft and toward the PAZ lobe. Functional analyses reveal that this lysine promotes target binding and cleavage. Our study therefore provides a molecular basis for the piRNA targeting mechanism in mice and humans, and suggests that mammalian piRNA machinery can achieve broad target silencing using a limited supply of piRNA species.
History
DepositionJul 8, 2022-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateMay 8, 2024-
Current statusMay 8, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33797.png

Downloads & links

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Map

FileDownload / File: emd_33797.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationoverall map
Voxel sizeX=Y=Z: 1.087 Å
Density
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-4.7239294 - 6.6546907
Average (Standard dev.)0.00094602077 (±0.16118972)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 208.70401 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: DeepEMhancer processed map

Fileemd_33797_additional_1.map
AnnotationDeepEMhancer processed map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_33797_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_33797_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EfPiwi(N959K)-piRNA-16nt target

EntireName: EfPiwi(N959K)-piRNA-16nt target
Components
  • Complex: EfPiwi(N959K)-piRNA-16nt target
    • Complex: EfPiwi(N959K)
      • Protein or peptide: Piwi
    • Complex: RNA
      • RNA: piRNA
      • RNA: RNA (5'-R(*UP*CP*CP*AP*UP*GP*UP*UP*GP*AP*UP*GP*GP*UP*AP*A)-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: EfPiwi(N959K)-piRNA-16nt target

SupramoleculeName: EfPiwi(N959K)-piRNA-16nt target / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: EfPiwi(N959K)

SupramoleculeName: EfPiwi(N959K) / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Ephydatia fluviatilis (invertebrata)

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Supramolecule #3: RNA

SupramoleculeName: RNA / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2

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Macromolecule #1: piRNA

MacromoleculeName: piRNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 7.957792 KDa
SequenceString:
UUACCAUCAA CAUGGAAACU UGGC(OMU)

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Macromolecule #2: RNA (5'-R(*UP*CP*CP*AP*UP*GP*UP*UP*GP*AP*UP*GP*GP*UP*AP*A)-3')

MacromoleculeName: RNA (5'-R(*UP*CP*CP*AP*UP*GP*UP*UP*GP*AP*UP*GP*GP*UP*AP*A)-3')
type: rna / ID: 2 / Details: 16nt RNA target / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 5.100047 KDa
SequenceString:
UCCAUGUUGA UGGUAA

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Macromolecule #3: Piwi

MacromoleculeName: Piwi / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Ephydatia fluviatilis (invertebrata)
Molecular weightTheoretical: 92.654094 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MDYKDDDDKG SDYKDDDDKG SDYKDDDDKG SENLYFQGSL NVESSMVSQR GSSGQPVPVS ANYLPLKGNM DGVFKYAVGF NPPVEDIRS RSQLLNEHKE LIGLTRVFDG STLYVPKRIC EQRLDLMSTR QTDGASIKVT ISLVDSVKNR DVVQLMNVIF K RILRSLKL ...String:
MDYKDDDDKG SDYKDDDDKG SDYKDDDDKG SENLYFQGSL NVESSMVSQR GSSGQPVPVS ANYLPLKGNM DGVFKYAVGF NPPVEDIRS RSQLLNEHKE LIGLTRVFDG STLYVPKRIC EQRLDLMSTR QTDGASIKVT ISLVDSVKNR DVVQLMNVIF K RILRSLKL QRIGRDYYDA NSPLEVPQHK MQLWPGYVTA INRHEGGLML VLDVSHRVMK TDTALDFLYE LYHFNQDKFR EE AFKQLVG SVVLTRYNNR TYEIDDIAWD KNPRCAFQDH AGSQITFVDY YKRAYDLDIT DLEQPLLIHR PKKKQRGKQD EGR KEVEEM VCLVPELCAM TGLTDAARSD FKVMKDLAVH TRVPPEKRAE SFRKFIQRLN TTKEASELLH SWGLVLDSRM LDMQ GRRLP PEKILFKHSS IVANMEADWS RECLKEHVIS AVSLLDWAVL FVRKDQGKAT DFVNMLSKVC PPIGMEVHEP KMVEV VNDR TESYLRALRE LIAPRLQMVV IVFPTSRDDR YSAVKKLCCI ESPIPSQVLI ARTITQQQKL RSVAQKVALQ MNAKLG GEL WAVEIPLKSC MVVGIDVYHD KSYGNKSIAG FVASTNPSFT RWYSRTAMQE QSQELIHELK LCMQAALKKY NEMNQSL PE RIIVFRDGVG EGREEYVSEF EVPQFNSCFS IFGENYCPKL AVVVVQKRIT TRIFGRSGHS YDNPPPGVIV DHTITKSY D FYLVSQHVRQ GTVSPTYYRV IYDKSGLKPD HLQRLTYKLT HMYYNWPGTI RTPAPCKYAH KLAFLVGKSL HRDPAHELS DRLFFL

UniProtKB: Piwi

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / 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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 221896

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