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- EMDB-22190: Subtomogram averaging map of yeast polysome -

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Basic information

Entry
Database: EMDB / ID: EMD-22190
TitleSubtomogram averaging map of yeast polysome
Map data
Sample
  • Complex: Yeast Ribosome
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodelectron tomography / cryo EM
AuthorsKastritis PL
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health Sciences (NIH/NIEHS)5R00ES025835-05 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2020
Title: Structural impact of K63 ubiquitin on yeast translocating ribosomes under oxidative stress.
Authors: Ye Zhou / Panagiotis L Kastritis / Shannon E Dougherty / Jonathan Bouvette / Allen L Hsu / Laura Burbaum / Shyamal Mosalaganti / Stefan Pfeffer / Wim J H Hagen / Friedrich Förster / Mario J ...Authors: Ye Zhou / Panagiotis L Kastritis / Shannon E Dougherty / Jonathan Bouvette / Allen L Hsu / Laura Burbaum / Shyamal Mosalaganti / Stefan Pfeffer / Wim J H Hagen / Friedrich Förster / Mario J Borgnia / Christine Vogel / Martin Beck / Alberto Bartesaghi / Gustavo M Silva /
Abstract: Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational ...Subpopulations of ribosomes are responsible for fine tuning the control of protein synthesis in dynamic environments. K63 ubiquitination of ribosomes has emerged as a new posttranslational modification that regulates protein synthesis during cellular response to oxidative stress. K63 ubiquitin, a type of ubiquitin chain that functions independently of the proteasome, modifies several sites at the surface of the ribosome, however, we lack a molecular understanding on how this modification affects ribosome structure and function. Using cryoelectron microscopy (cryo-EM), we resolved the first three-dimensional (3D) structures of K63 ubiquitinated ribosomes from oxidatively stressed yeast cells at 3.5-3.2 Å resolution. We found that K63 ubiquitinated ribosomes are also present in a polysome arrangement, similar to that observed in yeast polysomes, which we determined using cryoelectron tomography (cryo-ET). We further showed that K63 ubiquitinated ribosomes are captured uniquely at the rotated pretranslocation stage of translation elongation. In contrast, cryo-EM structures of ribosomes from mutant cells lacking K63 ubiquitin resolved at 4.4-2.7 Å showed 80S ribosomes represented in multiple states of translation, suggesting that K63 ubiquitin regulates protein synthesis at a selective stage of elongation. Among the observed structural changes, ubiquitin mediates the destabilization of proteins in the 60S P-stalk and in the 40S beak, two binding regions of the eukaryotic elongation factor eEF2. These changes would impact eEF2 function, thus, inhibiting translocation. Our findings help uncover the molecular effects of K63 ubiquitination on ribosomes, providing a model of translation control during oxidative stress, which supports elongation halt at pretranslocation.
History
DepositionJun 19, 2020-
Header (metadata) releaseAug 26, 2020-
Map releaseAug 26, 2020-
UpdateSep 8, 2021-
Current statusSep 8, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22190.png

Downloads & links

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Map

FileDownload / File: emd_22190.map.gz / Format: CCP4 / Size: 1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 5.24 Å
Density
Contour LevelMovie #1: 1
Minimum - Maximum-4.829763 - 10.962913
Average (Standard dev.)9.856297e-08 (±0.999995)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions646464
Spacing646464
CellA=B=C: 335.36 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.245.245.24
M x/y/z646464
origin x/y/z0.0000.0000.000
length x/y/z335.360335.360335.360
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ300300300
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS646464
D min/max/mean-4.83010.9630.000

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Supplemental data

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Sample components

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Entire : Yeast Ribosome

EntireName: Yeast Ribosome
Components
  • Complex: Yeast Ribosome

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Supramolecule #1: Yeast Ribosome

SupramoleculeName: Yeast Ribosome / 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
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4
Details: 20 mM HEPES, pH 7.4, 10 mM MgCl2, 150 mM KCl, 1 mM DTT, 0.1 mg/mL cycloheximide
GridDetails: unspecified
VitrificationCryogen name: ETHANE
SectioningOther: NO SECTIONING
Fiducial markerManufacturer: Sigma-Aldrich / Diameter: 10 nm

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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
Image recordingFilm or detector model: GATAN K2 BASE (4k x 4k) / Average electron dose: 100.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionNumber images used: 60

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