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- EMDB-22040: Asterix/Gtsf1 from mouse (full-length protein) bound to co-purify... -

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Basic information

Entry
Database: EMDB / ID: EMD-22040
TitleAsterix/Gtsf1 from mouse (full-length protein) bound to co-purifying tRNA
Map dataAsterix/Gtsf1 from mouse (full-length protein) bound to co-purifying tRNA
Sample
  • Complex: Complex of Asterix/Gtsf1 from mouse (full-length protein) bound to co-purifying tRNA
    • Complex: Gametocyte-specific factor 1
      • Protein or peptide: Gametocyte-specific factor 1
    • Cell: tRNA (heterogeneous population)
      • RNA: tRNA
Biological speciesMus musculus (house mouse) / Spodoptera frugiperda (fall armyworm)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.8 Å
AuthorsIpsaro JJ / Joshua-Tor L
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)F32GM097888 United States
CitationJournal: Cell Rep / Year: 2021
Title: Asterix/Gtsf1 links tRNAs and piRNA silencing of retrotransposons.
Authors: Jonathan J Ipsaro / Paul A O'Brien / Shibani Bhattacharya / Arthur G Palmer / Leemor Joshua-Tor /
Abstract: The Piwi-interacting RNA (piRNA) pathway safeguards genomic integrity by silencing transposable elements (transposons) in the germline. While Piwi is the central piRNA factor, others including ...The Piwi-interacting RNA (piRNA) pathway safeguards genomic integrity by silencing transposable elements (transposons) in the germline. While Piwi is the central piRNA factor, others including Asterix/Gtsf1 have also been demonstrated to be critical for effective silencing. Here, using enhanced crosslinking and immunoprecipitation (eCLIP) with a custom informatic pipeline, we show that Asterix/Gtsf1 specifically binds tRNAs in cellular contexts. We determined the structure of mouse Gtsf1 by NMR spectroscopy and identified the RNA-binding interface on the protein's first zinc finger, which was corroborated by biochemical analysis as well as cryo-EM structures of Gtsf1 in complex with co-purifying tRNA. Consistent with the known dependence of long terminal repeat (LTR) retrotransposons on tRNA primers, we demonstrate that LTR retrotransposons are, in fact, preferentially de-repressed in Asterix mutants. Together, these findings link Asterix/Gtsf1, tRNAs, and LTR retrotransposon silencing and suggest that Asterix exploits tRNA dependence to identify transposon transcripts and promote piRNA silencing.
History
DepositionMay 22, 2020-
Header (metadata) releaseMar 3, 2021-
Map releaseMar 3, 2021-
UpdateApr 14, 2021-
Current statusApr 14, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22040.png

Downloads & links

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Map

FileDownload / File: emd_22040.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsterix/Gtsf1 from mouse (full-length protein) bound to co-purifying tRNA
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.63 Å/pix.
x 300 pix.
= 187.86 Å		0.63 Å/pix.
x 300 pix.
= 187.86 Å		0.63 Å/pix.
x 300 pix.
= 187.86 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.6262 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum0.017668411 - 1.5232673
Average (Standard dev.)0.04056054 (±0.08181212)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 187.86 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.62620.62620.6262
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z187.860187.860187.860
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean0.0181.5230.041

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Supplemental data

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Sample components

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Entire : Complex of Asterix/Gtsf1 from mouse (full-length protein) bound t...

EntireName: Complex of Asterix/Gtsf1 from mouse (full-length protein) bound to co-purifying tRNA
Components
  • Complex: Complex of Asterix/Gtsf1 from mouse (full-length protein) bound to co-purifying tRNA
    • Complex: Gametocyte-specific factor 1
      • Protein or peptide: Gametocyte-specific factor 1
    • Cell: tRNA (heterogeneous population)
      • RNA: tRNA

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Supramolecule #1: Complex of Asterix/Gtsf1 from mouse (full-length protein) bound t...

SupramoleculeName: Complex of Asterix/Gtsf1 from mouse (full-length protein) bound to co-purifying tRNA
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Co-purifying RNA in this sample was derived from the expression host (Sf9) and represents a heterogenous population. For more details on the identities of these nucleic acids, please see the ...Details: Co-purifying RNA in this sample was derived from the expression host (Sf9) and represents a heterogenous population. For more details on the identities of these nucleic acids, please see the original publication.
Molecular weightTheoretical: 45 KDa

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Supramolecule #2: Gametocyte-specific factor 1

SupramoleculeName: Gametocyte-specific factor 1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Supramolecule #3: tRNA (heterogeneous population)

SupramoleculeName: tRNA (heterogeneous population) / type: cell / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Gametocyte-specific factor 1

MacromoleculeName: Gametocyte-specific factor 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MEDTYIDSLD PEKLLQCPYD KNHQIRACRF PYHLIKCRKN HPDVANKLAT CPFNARHQVP RAEISHHISS CDDKSCIEQD VVNQTRNLGQ ETLAESTWQC PPCDEDWDKD LWEQTSTPFV WGTASFCGNN SPANNIVMEH KSNLASGMRV PKSLPYVLPW KNNGNAQENL ...String:
MEDTYIDSLD PEKLLQCPYD KNHQIRACRF PYHLIKCRKN HPDVANKLAT CPFNARHQVP RAEISHHISS CDDKSCIEQD VVNQTRNLGQ ETLAESTWQC PPCDEDWDKD LWEQTSTPFV WGTASFCGNN SPANNIVMEH KSNLASGMRV PKSLPYVLPW KNNGNAQENL YFQGASAWSH PQFEKGGGSG GGSGGSAWSH PQFEK

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Macromolecule #2: tRNA

MacromoleculeName: tRNA / type: rna / ID: 2
Details: The sequence above represents the most abundant RNA as assessed by next-generation sequencing. This sequence made up approximately 15% of the sequencing library, with additional sequences ...Details: The sequence above represents the most abundant RNA as assessed by next-generation sequencing. This sequence made up approximately 15% of the sequencing library, with additional sequences only 1 nucleotide different making up approximately 10% more of the library.
Source (natural)Organism: Spodoptera frugiperda (fall armyworm) / Tissue: ovary
SequenceString:
TCTTCGGTAG TATAGTGGTC AGTATCCCCG CCTGTCACGC GGGAGACCGG GGTTCGATTC CCCGCCGGAG AGCCA

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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

Concentration0.25 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
100.0 mMKClpotassium chloride
5.0 mMC10H18N2O3d-desthiobiotin
GridModel: Homemade / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: LACEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 4.0 kPa
Details: Grids were purchased from Electron Microscopy Sciences. Lacey carbon film, 300 mesh.
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 298 K / Instrument: LEICA EM GP / Details: Blotted for 2.5 seconds before plunging..
DetailsMonodisperse complex with an apparent stoichometry of 1:1 protein:RNA based on gel filtration.

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4849 / Average exposure time: 4.0 sec. / Average electron dose: 76.4 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 215000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 495299
CTF correctionSoftware - Name: Warp (ver. 1.0.6)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 9.8 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: cisTEM (ver. 1.0.0)
Details: The resolution estimate by FSC is likely higher than the actual model. Visual inspection of the map suggests a resolution closer to 15-20 Angstroms.
Number images used: 346643
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM (ver. 1.0.0-beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM (ver. 1.0.0-beta)
Final 3D classificationNumber classes: 17 / Software - Name: cisTEM (ver. 1.0.0-beta)
FSC plot (resolution estimation)

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