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- EMDB-15810: In situ Dictyostelium discoideum 80S ribosome with eEF1A, A/T-tRN... -

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Basic information

Entry
Database: EMDB / ID: EMD-15810
TitleIn situ Dictyostelium discoideum 80S ribosome with eEF1A, A/T-tRNA and P-tRNA bound
Map dataSubtomogram average of 80S Dictyostelium discoideum ribosome with eEF1A, A/T-tRNA and P-tRNA bound
Sample
  • Complex: 80S ribosome from inside Dictyostelium discoideum cells.
Keywordseukaryotic ribosome / translation / tRNA / elongation factor / expansion segments / eEF1A / RIBOSOME
Biological speciesDictyostelium discoideum AX2 (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 6.9 Å
AuthorsHoffmann PC / Kreysing JP / Khusainov I / Tuijtel MW / Welsch S / Beck M
Funding support Germany, United States, 2 items
OrganizationGrant numberCountry
Max Planck Society Germany
Chan Zuckerberg Initiative United States
CitationJournal: Nat Commun / Year: 2022
Title: Structures of the eukaryotic ribosome and its translational states in situ.
Authors: Patrick C Hoffmann / Jan Philipp Kreysing / Iskander Khusainov / Maarten W Tuijtel / Sonja Welsch / Martin Beck /
Abstract: Ribosomes translate genetic information into primary structure. During translation, various cofactors transiently bind to the ribosome that undergoes prominent conformational and structural changes. ...Ribosomes translate genetic information into primary structure. During translation, various cofactors transiently bind to the ribosome that undergoes prominent conformational and structural changes. Different translational states of ribosomes have been well characterized in vitro. However, to which extent the known translational states are representative of the native situation inside cells has thus far only been addressed in prokaryotes. Here, we apply cryo-electron tomography to cryo-FIB milled Dictyostelium discoideum cells combined with subtomogram averaging and classification. We obtain an in situ structure that is locally resolved up to 3 Angstrom, the distribution of eukaryotic ribosome translational states, and unique arrangement of rRNA expansion segments. Our work demonstrates the use of in situ structural biology techniques for identifying distinct ribosome states within the cellular environment.
History
DepositionSep 14, 2022-
Header (metadata) releaseDec 7, 2022-
Map releaseDec 7, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15810.png

Downloads & links

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Map

FileDownload / File: emd_15810.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomogram average of 80S Dictyostelium discoideum ribosome with eEF1A, A/T-tRNA and P-tRNA bound
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.39 Å/pix.
x 180 pix.
= 430.848 Å		2.39 Å/pix.
x 180 pix.
= 430.848 Å		2.39 Å/pix.
x 180 pix.
= 430.848 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.3936 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-1.0594859 - 1.9898404
Average (Standard dev.)0.037514992 (±0.16323806)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 430.848 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Subtomogram average of 80S Dictyostelium discoideum ribosome with...

Fileemd_15810_half_map_1.map
AnnotationSubtomogram average of 80S Dictyostelium discoideum ribosome with eEF1A, A/T-tRNA and P-tRNA bound
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Subtomogram average of 80S Dictyostelium discoideum ribosome with...

Fileemd_15810_half_map_2.map
AnnotationSubtomogram average of 80S Dictyostelium discoideum ribosome with eEF1A, A/T-tRNA and P-tRNA bound
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : 80S ribosome from inside Dictyostelium discoideum cells.

EntireName: 80S ribosome from inside Dictyostelium discoideum cells.
Components
  • Complex: 80S ribosome from inside Dictyostelium discoideum cells.

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Supramolecule #1: 80S ribosome from inside Dictyostelium discoideum cells.

SupramoleculeName: 80S ribosome from inside Dictyostelium discoideum cells.
type: complex / ID: 1 / Parent: 0
Details: Cells from axenic Dictyostelium discoideum strain Ax2-214 with integrated GFP-Nup62 grown in HL5 medium were plunge frozen on Au 200mesh EM support grids.
Source (natural)Organism: Dictyostelium discoideum AX2 (eukaryote) / Strain: Ax2-214 / Location in cell: cytoplasm of perinuclear region
Molecular weightTheoretical: 4.2 MDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 6.5
GridModel: Quantifoil / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber temperature: 294 K / Instrument: LEICA EM GP

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 2.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.5 µm / Nominal magnification: 42000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number subtomograms used: 8939
ExtractionNumber tomograms: 127 / Number images used: 127000
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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