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Open data
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Basic information
Entry | Database: PDB / ID: 7kx7 | ||||||
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Title | Cryo-EM structure of Ephydatia fluviatilis PiwiA-piRNA complex | ||||||
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![]() | HYDROLASE / RNA BINDING PROTEIN/RNA / RNASEH / PROTEIN-RNA COMPLEX / RNA BINDING PROTEIN / PIRNA BINDING / PAZ PIWI DOMAIN PROTEIN / RNA BINDING PROTEIN-RNA complex | ||||||
Function / homology | ![]() piRNA binding / regulatory ncRNA-mediated gene silencing / P granule / RNA endonuclease activity Similarity search - Function | ||||||
Biological species | ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å | ||||||
![]() | Anzelon, T.A. / Chowdhury, S. / Lander, G.C. / MacRae, I.J. | ||||||
Funding support | ![]()
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![]() | ![]() 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. | ||||||
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Structure visualization
Movie |
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 148.2 KB | Display | ![]() |
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PDB format | ![]() | 110.4 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 23061MC ![]() 7kx9C M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | 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.) ![]() ![]() ![]() References: UniProt: D5MRY8, Hydrolases; Acting on ester bonds; Endoribonucleases producing 5'-phosphomonoesters | ||||
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#2: RNA chain | Mass: 8029.815 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) | ||||
#3: Chemical | Compound details | Gaps in the structure correspond to regions lacking density, that could not be confidently modeled | Has ligand of interest | N | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
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Molecular weight | Value: 0.083 MDa / Experimental value: NO | ||||||||||||||||||||||||
Source (natural) |
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Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||
Specimen | Conc.: 2.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||||||||||||||
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / 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 holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 79 K / Temperature (min): 77 K |
Image recording | Average 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 scans | Sampling size: 5 µm / Width: 3710 / Height: 3838 / Movie frames/image: 48 / Used frames/image: 1-48 |
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Processing
Software | Name: PHENIX / Version: 1.18.2_3874: / Classification: refinement | |||||||||||||||||||||||||||||||||||
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EM software |
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Image processing | Details: 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 correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | |||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 3280351 Details: Laplacian of Gaussian based automated particle picking program in RELION was used. | |||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | |||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1862936 / Algorithm: BACK PROJECTION / Symmetry type: POINT |