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- EMDB-15215: Asymmetric reconstruction of averaged ribosomes from Saccharomyce... -

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Basic information

Entry
Database: EMDB / ID: EMD-15215
TitleAsymmetric reconstruction of averaged ribosomes from Saccharomyces cerevisiae
Map dataAsymmetric reconstruction of averaged ribosomes from Saccharomyces cerevisiae
Sample
  • Complex: Saccharomyces cerevisiae ribosomes
Keywordsribosome / replication / active translation / ribosomal states / polysomes
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.1 Å
AuthorsSchmidt L / Tueting C / Kyrilis F / Hamdi F / Semchonok DA / Kastritis PL
Funding support Germany, European Union, 5 items
OrganizationGrant numberCountry
German Federal Ministry for Education and Research Germany
German Federal Ministry for Education and Research03Z22HI2 Germany
German Federal Ministry for Education and Research03COV04 Germany
German Research Foundation (DFG)391498659 Germany
European Regional Development FundEFRE: ZS/2016/04/78115European Union
Citation
Journal: Commun Biol / Year: 2024
Title: Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts.
Authors: Lisa Schmidt / Christian Tüting / Fotis L Kyrilis / Farzad Hamdi / Dmitry A Semchonok / Gerd Hause / Annette Meister / Christian Ihling / Milton T Stubbs / Andrea Sinz / Panagiotis L Kastritis /
Abstract: The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to ...The high abundance of most viruses in infected host cells benefits their structural characterization. However, endogenous viruses are present in low copy numbers and are therefore challenging to investigate. Here, we retrieve cell extracts enriched with an endogenous virus, the yeast L-A virus. The determined cryo-EM structure discloses capsid-stabilizing cation-π stacking, widespread across viruses and within the Totiviridae, and an interplay of non-covalent interactions from ten distinct capsomere interfaces. The capsid-embedded mRNA decapping active site trench is supported by a constricting movement of two flexible opposite-facing loops. tRNA-loaded polysomes and other biomacromolecules, presumably mRNA, are found in virus proximity within the cell extract. Mature viruses participate in larger viral communities resembling their rare in-cell equivalents in terms of size, composition, and inter-virus distances. Our results collectively describe a 3D-architecture of a viral milieu, opening the door to cell-extract-based high-resolution structural virology.
#1: Journal: Biorxiv / Year: 2022
Title: Delineating organizational principles of the endogenous L-A virus by cryo-EM and computational analysis of native cell extracts
Authors: Schmidt L / Tuting C / Kyrilis FL / Hamdi F / Semchonok DA / Hause G / Meister A / Ihling C / Shah PNM / Stubbs MT / Sinz A / Stuart DI / Kastritis PL
History
DepositionJun 20, 2022-
Header (metadata) releaseDec 20, 2023-
Map releaseDec 20, 2023-
UpdateJun 19, 2024-
Current statusJun 19, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15215.png

Downloads & links

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Map

FileDownload / File: emd_15215.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsymmetric reconstruction of averaged ribosomes from Saccharomyces cerevisiae
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
3.18 Å/pix.
x 128 pix.
= 406.656 Å		3.18 Å/pix.
x 128 pix.
= 406.656 Å		3.18 Å/pix.
x 128 pix.
= 406.656 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3.177 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 2.74
Minimum - Maximum-1.183511 - 4.262439
Average (Standard dev.)0.14208034 (±0.61363304)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 406.656 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Reconstructed map of a ribosome after variability analysis...

Fileemd_15215_additional_1.map
AnnotationReconstructed map of a ribosome after variability analysis showing tRNA occupation at sites A/A P/P
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Additional map: Reconstructed map of a ribosome after variability analysis...

Fileemd_15215_additional_2.map
AnnotationReconstructed map of a ribosome after variability analysis showing tRNA occupation at sites P/P E/E
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Additional map: Reconstructed map of a ribosome after variability analysis...

Fileemd_15215_additional_3.map
AnnotationReconstructed map of a ribosome after variability analysis showing tRNA occupation at sites P/E E/-
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Additional map: Reconstructed map of a ribosome after variability analysis...

Fileemd_15215_additional_4.map
AnnotationReconstructed map of a ribosome after variability analysis showing tRNA occupation at sites A/P P/E
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Additional map: Reconstructed map of a ribosome after variability analysis...

Fileemd_15215_additional_5.map
AnnotationReconstructed map of a ribosome after variability analysis showing tRNA occupation at site A/P
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Additional map: Reconstructed map of a ribosome after variability analysis...

Fileemd_15215_additional_6.map
AnnotationReconstructed map of a ribosome after variability analysis showing tRNA occupation at site A/A
Projections & Slices
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Half map: Asymmetric reconstruction of averaged ribosomes from Saccharomyces cerevisiae...

Fileemd_15215_half_map_1.map
AnnotationAsymmetric reconstruction of averaged ribosomes from Saccharomyces cerevisiae - Half Map B
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Half map: Asymmetric reconstruction of averaged ribosomes from Saccharomyces cerevisiae...

Fileemd_15215_half_map_2.map
AnnotationAsymmetric reconstruction of averaged ribosomes from Saccharomyces cerevisiae - Half Map A
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Sample components

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Entire : Saccharomyces cerevisiae ribosomes

EntireName: Saccharomyces cerevisiae ribosomes
Components
  • Complex: Saccharomyces cerevisiae ribosomes

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Supramolecule #1: Saccharomyces cerevisiae ribosomes

SupramoleculeName: Saccharomyces cerevisiae ribosomes / type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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

Concentration0.3 mg/mL
BufferpH: 7.4 / Component - Concentration: 200.0 mM / Component - Formula: CH3COONH4 / Component - Name: Ammoniumacetate
Details: pH of the buffer was adjusted with NaOH buffer was filtered and sonicated
GridModel: Quantifoil R2/1 / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.04 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: blot force 2 and blot time 6 before plunging.
Detailsheterogenous cell extract

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Electron microscopy

MicroscopeTFS GLACIOS
TemperatureMin: 77.0 K / Max: 118.0 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 7 / Number real images: 2728 / Average exposure time: 3.61 sec. / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 44067 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 45000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 750242
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. v3.3.1) / Number images used: 627748
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. v3.3.1)
Final 3D classificationSoftware - Name: cryoSPARC (ver. v3.3.1)
FSC plot (resolution estimation)

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