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- EMDB-23063: Cryo-EM structure of Ephydatia fluviatilis PiwiA-piRNA-target complex -

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Basic information

Entry
Database: EMDB / ID: EMD-23063
TitleCryo-EM structure of Ephydatia fluviatilis PiwiA-piRNA-target complex
Map dataLocal resolution based filtered map of EfPiwi-piRNA-target complex.
Sample
  • Complex: EfPiwi-piRNA-target complex
    • Complex: Piwi-A
      • Protein or peptide: Piwi-A
    • Complex: piRNA-target
      • RNA: RNA (5'-R(P*UP*CP*UP*CP*UP*UP*GP*AP*GP*UP*UP*GP*GP*AP*CP*AP*AP*AP*UP*GP*GP*CP*AP*(OMG))-3')
      • RNA: RNA (5'-R(P*UP*GP*UP*CP*CP*AP*AP*CP*UP*CP*AP*AP*GP*AP*GP*A)-3')
  • Ligand: MAGNESIUM ION
KeywordsRNASEH / PROTEIN-RNA COMPLEX / RNA BINDING PROTEIN / PIRNA BINDING / PAZ PIWI DOMAIN PROTEIN / HYDROLASE / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


piRNA binding / regulatory ncRNA-mediated gene silencing / P granule / RNA endonuclease activity
Similarity search - Function
Piwi domain / Piwi domain profile. / 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: 3.5 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-
UpdateMay 29, 2024-
Current statusMay 29, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.055
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7kx9
  • Surface level: 0.055
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23063.png

Downloads & links

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Map

FileDownload / File: emd_23063.map.gz / Format: CCP4 / Size: 2.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLocal resolution based filtered map of EfPiwi-piRNA-target complex.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.15 Å/pix.
x 90 pix.
= 103.5 Å		1.15 Å/pix.
x 90 pix.
= 103.5 Å		1.15 Å/pix.
x 90 pix.
= 103.5 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: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.40370464 - 0.5853153
Average (Standard dev.)0.0032600898 (±0.032965515)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions909090
Spacing909090
CellA=B=C: 103.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.151.151.15
M x/y/z909090
origin x/y/z0.0000.0000.000
length x/y/z103.500103.500103.500
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS909090
D min/max/mean-0.4040.5850.003

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Supplemental data

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Mask #1

Fileemd_23063_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map of EfPiwi-piRNA-target complex.

Fileemd_23063_additional_1.map
AnnotationUnsharpened map of EfPiwi-piRNA-target complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Odd half map of EfPiwi-piRNA-target complex.

Fileemd_23063_half_map_1.map
AnnotationOdd half map of EfPiwi-piRNA-target complex.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Even half map of EfPiwi-piRNA-target complex.

Fileemd_23063_half_map_2.map
AnnotationEven half map of EfPiwi-piRNA-target complex.
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-piRNA-target complex

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

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Supramolecule #1: EfPiwi-piRNA-target complex

SupramoleculeName: EfPiwi-piRNA-target complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 94 KDa

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Supramolecule #2: Piwi-A

SupramoleculeName: Piwi-A / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Ephydatia fluviatilis (invertebrata)

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Supramolecule #3: piRNA-target

SupramoleculeName: piRNA-target / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: synthetic construct (others) / Synthetically produced: Yes

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Macromolecule #1: Piwi-A

MacromoleculeName: Piwi-A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Ephydatia fluviatilis (invertebrata)
Molecular weightTheoretical: 88.232594 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: SLNVESSMVS QRGSSGQPVP VSANYLPLKG NMDGVFKYAV GFNPPVEDIR SRSQLLNEHK ELIGLTRVFD GSTLYVPKRI CEQRLDLMS TRQTDGASIK VTISLVDSVK NRDVVQLMNV IFKRILRSLK LQRIGRDYYD ANSPLEVPQH KMQLWPGYVT A INRHEGGL ...String:
SLNVESSMVS QRGSSGQPVP VSANYLPLKG NMDGVFKYAV GFNPPVEDIR SRSQLLNEHK ELIGLTRVFD GSTLYVPKRI CEQRLDLMS TRQTDGASIK VTISLVDSVK NRDVVQLMNV IFKRILRSLK LQRIGRDYYD ANSPLEVPQH KMQLWPGYVT A INRHEGGL MLVLDVSHRV MKTDTALDFL YELYHFNQDK FREEAFKQLV GSVVLTRYNN RTYEIDDIAW DKNPRCAFQD HA GSQITFV DYYKRAYDLD ITDLEQPLLI HRPKKKQRGK QDEGRKEVEE MVCLVPELCA MTGLTDAARS DFKVMKDLAV HTR VPPEKR AESFRKFIQR LNTTKEASEL LHSWGLVLDS RMLDMQGRRL PPEKILFKHS SIVANMEADW SRECLKEHVI SAVS LLDWA VLFVRKDQGK ATDFVNMLSK VCPPIGMEVH EPKMVEVVND RTESYLRALR ELIAPRLQMV VIVFPTSRDD RYSAV KKLC CIESPIPSQV LIARTITQQQ KLRSVAQKVA LQMNAKLGGE LWAVEIPLKS CMVVGIDVYH DKSYGNKSIA GFVAST NPS FTRWYSRTAM QEQSQELIHE LKLCMQAALK KYNEMNQSLP ERIIVFRDGV GEGREEYVSE FEVPQFNSCF SIFGENY CP KLAVVVVQKR ITTRIFGRSG HSYDNPPPGV IVDHTITKSY DFYLVSQHVR QGTVSPTYYR VIYDKSGLKP DHLQRLTY K LTHMYYNWPG TIRTPAPCNY AHKLAFLVGK SLHRDPAHEL SDRLFFL

UniProtKB: Piwi

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Macromolecule #2: RNA (5'-R(P*UP*CP*UP*CP*UP*UP*GP*AP*GP*UP*UP*GP*GP*AP*CP*AP*AP*AP...

MacromoleculeName: RNA (5'-R(P*UP*CP*UP*CP*UP*UP*GP*AP*GP*UP*UP*GP*GP*AP*CP*AP*AP*AP*UP*GP*GP*CP*AP*(OMG))-3')
type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 7.724633 KDa
SequenceString:
UCUCUUGAGU UGGACAAAUG GCA(OMG)

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

MacromoleculeName: RNA (5'-R(P*UP*GP*UP*CP*CP*AP*AP*CP*UP*CP*AP*AP*GP*AP*GP*A)-3')
type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.10512 KDa
SequenceString:
UGUCCAACUC AAGAGA

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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

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 / Pretreatment - Time: 7 sec.
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 - Frames/image: 1-64 / Number real images: 1881 / Average exposure time: 16.0 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: 2551046
Details: Laplacian of Gaussian based automated particle picking program in RELION was used.
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

5guh


Details: 40 Angstrom low pass filtered map was generated from Siwi crystal structure (PDB ID 5GUH) using the molmap function in UCSF Chimera.
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.0) / Number images used: 118493
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 2)
FSC plot (resolution estimation)

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