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- EMDB-13115: Cryo-EM structure of the 80S rabbit ribosome from reticulocyte lysate -

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Basic information

Entry
Database: EMDB / ID: EMD-13115
TitleCryo-EM structure of the 80S rabbit ribosome from reticulocyte lysate
Map data
Sample
  • Complex: Rabbit 80S ribosome from reticulocyte lysate
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsLeesch F / Lorenzo-Orts L / Grishkovskaya I / Kandolf S / Belacic K / Meinhart A / Haselbach D / Pauli A
Funding support Austria, Switzerland, 5 items
OrganizationGrant numberCountry
Austrian Research Promotion AgencyFFG-852936 Austria
Austrian Science FundSTART Projekt Y 1031-B28 Austria
Austrian Science FundSFB RNA-Deco F 80 Austria
Swiss National Science FoundationP2GEP3_191204 Switzerland
European Molecular Biology Organization (EMBO)ALTF 1165-2019
CitationJournal: Nature / Year: 2023
Title: A molecular network of conserved factors keeps ribosomes dormant in the egg.
Authors: Friederike Leesch / Laura Lorenzo-Orts / Carina Pribitzer / Irina Grishkovskaya / Josef Roehsner / Anastasia Chugunova / Manuel Matzinger / Elisabeth Roitinger / Katarina Belačić / Susanne ...Authors: Friederike Leesch / Laura Lorenzo-Orts / Carina Pribitzer / Irina Grishkovskaya / Josef Roehsner / Anastasia Chugunova / Manuel Matzinger / Elisabeth Roitinger / Katarina Belačić / Susanne Kandolf / Tzi-Yang Lin / Karl Mechtler / Anton Meinhart / David Haselbach / Andrea Pauli /
Abstract: Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed. Here, using mass spectrometry and cryo-electron ...Ribosomes are produced in large quantities during oogenesis and are stored in the egg. However, the egg and early embryo are translationally repressed. Here, using mass spectrometry and cryo-electron microscopy analyses of ribosomes isolated from zebrafish (Danio rerio) and Xenopus laevis eggs and embryos, we provide molecular evidence that ribosomes transition from a dormant state to an active state during the first hours of embryogenesis. Dormant ribosomes are associated with four conserved factors that form two modules, consisting of Habp4-eEF2 and death associated protein 1b (Dap1b) or Dap in complex with eIF5a. Both modules occupy functionally important sites and act together to stabilize ribosomes and repress translation. Dap1b (also known as Dapl1 in mammals) is a newly discovered translational inhibitor that stably inserts into the polypeptide exit tunnel. Addition of recombinant zebrafish Dap1b protein is sufficient to block translation and reconstitute the dormant egg ribosome state in a mammalian translation extract in vitro. Thus, a developmentally programmed, conserved ribosome state has a key role in ribosome storage and translational repression in the egg.
History
DepositionJun 24, 2021-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateMar 15, 2023-
Current statusMar 15, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_13115.png

Downloads & links

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Map

FileDownload / File: emd_13115.map.gz / Format: CCP4 / Size: 421.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.06 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.4425676 - 3.4857752
Average (Standard dev.)0.0051459074 (±0.07639814)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions480480480
Spacing480480480
CellA=B=C: 508.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Rabbit 80S ribosome from reticulocyte lysate

EntireName: Rabbit 80S ribosome from reticulocyte lysate
Components
  • Complex: Rabbit 80S ribosome from reticulocyte lysate

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Supramolecule #1: Rabbit 80S ribosome from reticulocyte lysate

SupramoleculeName: Rabbit 80S ribosome from reticulocyte lysate / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#83
Source (natural)Organism: Oryctolagus cuniculus (rabbit)

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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

BufferpH: 7.6
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
100.0 mMKClPotassium chloride
5.0 mMMg(OAc)2Magnesium acetate
1.0 mMDTTDithiothreitol
20.0 mMNH4ClAmmonium chloride
GridModel: Quantifoil R3.5/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 2.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7526 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
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: 1383919
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.0) / Number images used: 619214
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2.0)

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