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- EMDB-23062: Cryo-EM structure of EfPiwiA(MID/PIWI domains)-piRNA-long-target ... -

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Basic information

Entry
Database: EMDB / ID: EMD-23062
TitleCryo-EM structure of EfPiwiA(MID/PIWI domains)-piRNA-long-target complex
Map dataEfPiwi (MID/PIWI domains)-piRNA-long-target complex full map
Sample
  • Complex: EfPiwiA(MID/PIWI domains)-piRNA-long-target complex
    • Complex: PiwiA(MID/PIWI domains)
    • Complex: piRNA-long-target
Function / homology
Function and homology information


regulatory ncRNA-mediated gene silencing / cell differentiation / RNA binding / cytoplasm
Similarity search - Function
Piwi, N-terminal domain / Piwi domain profile. / Piwi domain / Piwi domain / Piwi / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain ...Piwi, N-terminal domain / Piwi domain profile. / Piwi domain / Piwi domain / Piwi / PAZ domain superfamily / PAZ / PAZ domain / PAZ domain profile. / PAZ domain / Ribonuclease H superfamily / Ribonuclease H-like superfamily
Similarity search - Domain/homology
Biological speciesEphydatia fluviatilis (invertebrata) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.6 Å
AuthorsChowdhury S / Anzelon TA / MacRae IJ / Lander GC
Funding support United States, 1 items
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, 2020-
Header (metadata) releaseJul 14, 2021-
Map releaseJul 14, 2021-
UpdateSep 22, 2021-
Current statusSep 22, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.324
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.324
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23062.png

Downloads & links

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Map

FileDownload / File: emd_23062.map.gz / Format: CCP4 / Size: 15.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEfPiwi (MID/PIWI domains)-piRNA-long-target complex full map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 160 pix.
= 184. Å		1.15 Å/pix.
x 160 pix.
= 184. Å		1.15 Å/pix.
x 160 pix.
= 184. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.15 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.324 / Movie #1: 0.324
Minimum - Maximum-0.93232226 - 1.8186889
Average (Standard dev.)-0.0015993412 (±0.07443944)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions160160160
Spacing160160160
CellA=B=C: 184.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z160160160
origin x/y/z0.0000.0000.000
length x/y/z184.000184.000184.000
α/β/γ90.00090.00090.000
start NX/NY/NZ10610885
NX/NY/NZ9087120
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS160160160
D min/max/mean-0.9321.819-0.002

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Supplemental data

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Mask #1

Fileemd_23062_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EfPiwi (MID/PIWI domains)-piRNA-long-target complex odd half map

Fileemd_23062_half_map_1.map
AnnotationEfPiwi (MID/PIWI domains)-piRNA-long-target complex odd half map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: EfPiwi (MID/PIWI domains)-piRNA-long-target complex even half map

Fileemd_23062_half_map_2.map
AnnotationEfPiwi (MID/PIWI domains)-piRNA-long-target complex even half 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 : EfPiwiA(MID/PIWI domains)-piRNA-long-target complex

EntireName: EfPiwiA(MID/PIWI domains)-piRNA-long-target complex
Components
  • Complex: EfPiwiA(MID/PIWI domains)-piRNA-long-target complex
    • Complex: PiwiA(MID/PIWI domains)
    • Complex: piRNA-long-target

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Supramolecule #1: EfPiwiA(MID/PIWI domains)-piRNA-long-target complex

SupramoleculeName: EfPiwiA(MID/PIWI domains)-piRNA-long-target complex / type: complex / ID: 1 / Parent: 0
Molecular weightTheoretical: 45 KDa

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Supramolecule #2: PiwiA(MID/PIWI domains)

SupramoleculeName: PiwiA(MID/PIWI domains) / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Ephydatia fluviatilis (invertebrata)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Supramolecule #3: piRNA-long-target

SupramoleculeName: piRNA-long-target / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: synthetic construct (others)

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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

Concentration2.5 mg/mL
BufferpH: 8
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: PLASMA CLEANING
VitrificationCryogen name: ETHANE / Chamber humidity: 97 % / Chamber temperature: 277.15 K / Instrument: HOMEMADE PLUNGER
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

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 77.0 K / Max: 79.0 K
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3710 pixel / Digitization - Dimensions - Height: 3838 pixel / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-48 / Number real images: 1765 / Average exposure time: 12.0 sec. / Average electron dose: 47.33 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 43478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.6 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 36000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

DetailsBeam-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.
Particle selectionNumber selected: 3280351
Details: Laplacian of Gaussian based automated particle picking program in RELION was used.
CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5guh


Details: Starting model was generated from ab-initio reconstruction in Cryosparc
Final reconstructionResolution.type: BY AUTHOR / Resolution: 8.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 116655
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION
FSC plot (resolution estimation)

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