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- EMDB-14216: Cryo-electron tomography of in vitro assembled Bruno oskar 3'UTR ... -

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Basic information

Entry
Database: EMDB / ID: EMD-14216
TitleCryo-electron tomography of in vitro assembled Bruno oskar 3'UTR RNA condensates aged for 30 minutes followed by addition of more oskar 3'UTR RNA
Map datatomogram of in vitro assembled Bruno oskar 3'UTR RNA condensates that were aged for 30 minutes followed by addition of more oskar 3'UTR RNA.
Sample
  • Complex: Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 400 nano molar oskar 3'UTR RNA
Biological speciesDrosophila melanogaster (fruit fly)
Methodelectron tomography / cryo EM
AuthorsBose M / Mahamid J
Funding support Germany, 1 items
OrganizationGrant numberCountry
EIPOD fellowship under Marie Sklodowska-Curie Actions COFUND Germany
CitationJournal: Cell / Year: 2022
Title: Liquid-to-solid phase transition of oskar ribonucleoprotein granules is essential for their function in Drosophila embryonic development.
Authors: Mainak Bose / Marko Lampe / Julia Mahamid / Anne Ephrussi /
Abstract: Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP ...Asymmetric localization of oskar ribonucleoprotein (RNP) granules to the oocyte posterior is crucial for abdominal patterning and germline formation in the Drosophila embryo. We show that oskar RNP granules in the oocyte are condensates with solid-like physical properties. Using purified oskar RNA and scaffold proteins Bruno and Hrp48, we confirm in vitro that oskar granules undergo a liquid-to-solid phase transition. Whereas the liquid phase allows RNA incorporation, the solid phase precludes incorporation of additional RNA while allowing RNA-dependent partitioning of client proteins. Genetic modification of scaffold granule proteins or tethering the intrinsically disordered region of human fused in sarcoma (FUS) to oskar mRNA allowed modulation of granule material properties in vivo. The resulting liquid-like properties impaired oskar localization and translation with severe consequences on embryonic development. Our study reflects how physiological phase transitions shape RNA-protein condensates to regulate the localization and expression of a maternal RNA that instructs germline formation.
History
DepositionJan 31, 2022-
Header (metadata) releaseFeb 23, 2022-
Map releaseFeb 23, 2022-
UpdateApr 27, 2022-
Current statusApr 27, 2022Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
  • Download
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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Movie viewer
Supplemental images

emd_14216.png

Downloads & links

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Map

FileDownload / File: emd_14216.map.gz / Format: CCP4 / Size: 2.5 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationtomogram of in vitro assembled Bruno oskar 3'UTR RNA condensates that were aged for 30 minutes followed by addition of more oskar 3'UTR RNA.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
8.51 Å/pix.
x 750 pix.
= 6382.5 Å		8.51 Å/pix.
x 928 pix.
= 7897.28 Å		8.51 Å/pix.
x 960 pix.
= 8169.6 Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 8.51 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-15.8751135 - 16.121902
Average (Standard dev.)0.22631682 (±2.2141483)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-1616375
Dimensions928960750
Spacing960928750
CellA: 8169.6 Å / B: 7897.2803 Å / C: 6382.5 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z8.518.518.51
M x/y/z960928750
origin x/y/z0.0000.0000.000
length x/y/z8169.6007897.2806382.500
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS16-16375
NC/NR/NS960928750
D min/max/mean-15.87516.1220.226

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Supplemental data

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Additional map: tomogram of in vitro assembled Bruno oskar 3'UTR...

Fileemd_14216_additional_1.map
Annotationtomogram of in vitro assembled Bruno oskar 3'UTR RNA condensates that were aged for 30 minutes followed by addition of more oskar 3'UTR RNA. Tomogram was gaussian filtered with a kernel size 3.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Condensates assembled using 10 micro molar Bruno-EGFP with 100 na...

EntireName: Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 400 nano molar oskar 3'UTR RNA
Components
  • Complex: Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 400 nano molar oskar 3'UTR RNA

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Supramolecule #1: Condensates assembled using 10 micro molar Bruno-EGFP with 100 na...

SupramoleculeName: Condensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 400 nano molar oskar 3'UTR RNA
type: complex / Chimera: Yes / ID: 1 / Parent: 0
Source (natural)Organism: Drosophila melanogaster (fruit fly)
Recombinant expressionOrganism: Spodoptera (butterflies/moths)

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Experimental details

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Structure determination

Methodcryo EM
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
VitrificationCryogen name: ETHANE-PROPANE
DetailsCondensates assembled using 10 micro molar Bruno-EGFP with 100 nano molar oskar 3'UTR RNA, aged for 30min followed by addition of 400 nano molar oskar 3'UTR RNA
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal defocus min: 3.0 µm
Specialist opticsPhase plate: VOLTA PHASE PLATE
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 2.1 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionSoftware - Name: IMOD / Number images used: 61

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