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- PDB-7kx7: Cryo-EM structure of Ephydatia fluviatilis PiwiA-piRNA complex -

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Basic information

Entry
Database: PDB / ID: 7kx7
TitleCryo-EM structure of Ephydatia fluviatilis PiwiA-piRNA complex
Components
  • Piwi-A
  • RNA (5'-R(P*UP*CP*UP*CP*AP*GP*(OMC))-3')
KeywordsHYDROLASE / RNA BINDING PROTEIN/RNA / RNASEH / PROTEIN-RNA COMPLEX / RNA BINDING PROTEIN / PIRNA BINDING / PAZ PIWI DOMAIN 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
RNA / RNA (> 10) / Piwi
Similarity search - Component
Biological speciesEphydatia fluviatilis (invertebrata)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsAnzelon, T.A. / Chowdhury, S. / Lander, G.C. / MacRae, I.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS) United States
CitationJournal: Nature / Year: 2021
Title: Structural basis for piRNA targeting.
Authors: Todd A Anzelon / Saikat Chowdhury / Siobhan M Hughes / Yao Xiao / Gabriel C Lander / Ian J MacRae /
Abstract: PIWI proteins use PIWI-interacting RNAs (piRNAs) to identify and silence transposable elements and thereby maintain genome integrity between metazoan generations. The targeting of transposable ...PIWI proteins use PIWI-interacting RNAs (piRNAs) to identify and silence transposable elements and thereby maintain genome integrity between metazoan generations. The targeting of transposable elements by PIWI has been compared to mRNA target recognition by Argonaute proteins, which use microRNA (miRNA) guides, but the extent to which piRNAs resemble miRNAs is not known. Here we present cryo-electron microscopy structures of a PIWI-piRNA complex from the sponge Ephydatia fluviatilis with and without target RNAs, and a biochemical analysis of target recognition. Mirroring Argonaute, PIWI identifies targets using the piRNA seed region. However, PIWI creates a much weaker seed so that stable target association requires further piRNA-target pairing, making piRNAs less promiscuous than miRNAs. Beyond the seed, the structure of PIWI facilitates piRNA-target pairing in a manner that is tolerant of mismatches, leading to long-lived PIWI-piRNA-target interactions that may accumulate on transposable-element transcripts. PIWI ensures targeting fidelity by physically blocking the propagation of piRNA-target interactions in the absence of faithful seed pairing, and by requiring an extended piRNA-target duplex to reach an endonucleolytically active conformation. PIWI proteins thereby minimize off-targeting cellular mRNAs while defending against evolving genomic threats.
History
DepositionDec 3, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 14, 2021Provider: repository / Type: Initial release
Revision 1.1Sep 1, 2021Group: Database references / Category: citation / database_2
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year / _database_2.pdbx_DOI / _database_2.pdbx_database_accession
Revision 1.2Sep 15, 2021Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Sep 22, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-23061
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Piwi-A
B: RNA (5'-R(P*UP*CP*UP*CP*AP*GP*(OMC))-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,3114
Polymers96,2622
Non-polymers492
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2800 Å2
ΔGint-35 kcal/mol
Surface area34380 Å2

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Components

#1: Protein Piwi-A / Piwi


Mass: 88232.594 Da / Num. of mol.: 1 / Mutation: N-terminal 219 amino acids deleted
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Ephydatia fluviatilis (invertebrata) / Gene: EfPiwiA / Production host: Spodoptera frugiperda (fall armyworm)
References: UniProt: D5MRY8, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters
#2: RNA chain RNA (5'-R(P*UP*CP*UP*CP*AP*GP*(OMC))-3')


Mass: 8029.815 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Compound detailsGaps in the structure correspond to regions lacking density, that could not be confidently modeled
Has ligand of interestN

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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

Component
IDNameTypeEntity IDParent-IDSource
1Complex between EfPiwi and piRNACOMPLEX#1-#20MULTIPLE SOURCES
2Piwi-ACOMPLEX#11RECOMBINANT
3piRNACOMPLEX#21SYNTHETIC
Molecular weightValue: 0.083 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Ephydatia fluviatilis (invertebrata)31330
33synthetic construct (others)32630
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
SpecimenConc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE / Humidity: 97 % / Chamber temperature: 277.15 K
Details: Freezing was carried out in a cold room at 4 degree C and relative humidity between 95%-98%. 3.5 uL sample was applied to plasma cleaned grid and manually blotted with Whatman 1 filter paper ...Details: Freezing was carried out in a cold room at 4 degree C and relative humidity between 95%-98%. 3.5 uL sample was applied to plasma cleaned grid and manually blotted with Whatman 1 filter paper for 5-7 sec before plunge freezing in liquid ethane at -179 degree C.

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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: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Calibrated magnification: 43478 X / Nominal defocus max: 1600 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79 K / Temperature (min): 77 K
Image recordingAverage exposure time: 12 sec. / Electron dose: 47.33 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1765
Image scansSampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 48 / Used frames/image: 1-48

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
1RELION2particle selection
2Leginonimage acquisitionData was collected using image shift-based movements from the center of four adjacent holes to target the center of each hole for exposures.
4CTFFIND4CTF correction
10RELION2initial Euler assignment
11RELION2final Euler assignment
13RELION23D reconstruction
Image processingDetails: Beam-induced motion correction and radiation damage compensation over spatial frequencies (dose-weighting) of the raw movies, was performed using UCSF MotionCor2 implemented in the Appion.
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3280351
Details: Laplacian of Gaussian based automated particle picking program in RELION was used.
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1862936 / Algorithm: BACK PROJECTION / Symmetry type: POINT

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