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- EMDB-15008: Subtomogram average of 80S ribosome from HeLa cell lysate -

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Basic information

Entry
Database: EMDB / ID: EMD-15008
TitleSubtomogram average of 80S ribosome from HeLa cell lysate
Map data
Sample
  • Complex: Polyribosomes from HeLa cytoplasm
Keywordseukaryotic polyribosome ribosomal translation complex / RIBOSOME
Biological speciesHomo sapiens (human)
Methodsubtomogram averaging / cryo EM / Resolution: 16.9 Å
AuthorsBaymukhametov TN / Chesnokov YM / Afonina ZA
Funding support Russian Federation, 1 items
OrganizationGrant numberCountry
Russian Foundation for Basic Research16-34-60148 Russian Federation
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Polyribosomes of circular topology are prevalent in mammalian cells.
Authors: Timur N Baymukhametov / Dmitry N Lyabin / Yury M Chesnokov / Ivan I Sorokin / Evgeniya V Pechnikova / Alexander L Vasiliev / Zhanna A Afonina /
Abstract: Polyribosomes, the groups of ribosomes simultaneously translating a single mRNA molecule, are very common in both, prokaryotic and eukaryotic cells. Even in early EM studies, polyribosomes have been ...Polyribosomes, the groups of ribosomes simultaneously translating a single mRNA molecule, are very common in both, prokaryotic and eukaryotic cells. Even in early EM studies, polyribosomes have been shown to possess various spatial conformations, including a ring-shaped configuration which was considered to be functionally important. However, a recent in situ cryo-ET analysis of predominant regular inter-ribosome contacts did not confirm the abundance of ring-shaped polyribosomes in a cell cytoplasm. To address this discrepancy, here we analyzed the cryo-ET structure of polyribosomes in diluted lysates of HeLa cells. It was shown that the vast majority of the ribosomes were combined into polysomes and were proven to be translationally active. Tomogram analysis revealed that circular polyribosomes are indeed very common in the cytoplasm, but they mostly possess pseudo-regular structures without specific inter-ribosomal contacts. Although the size of polyribosomes varied widely, most circular polysomes were relatively small in size (4-8 ribosomes). Our results confirm the recent data that it is cellular mRNAs with short ORF that most commonly form circular structures providing an enhancement of translation.
History
DepositionMay 20, 2022-
Header (metadata) releaseDec 21, 2022-
Map releaseDec 21, 2022-
UpdateDec 13, 2023-
Current statusDec 13, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15008.png

Downloads & links

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Map

FileDownload / File: emd_15008.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
3.7 Å/pix.
x 128 pix.
= 473.6 Å		3.7 Å/pix.
x 128 pix.
= 473.6 Å		3.7 Å/pix.
x 128 pix.
= 473.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 3.7 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.0
Minimum - Maximum-1.9039547 - 6.3372617
Average (Standard dev.)0.16693963 (±0.8574794)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 473.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_15008_msk_1.map
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AxesZYX

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Half map: #1

Fileemd_15008_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #2

Fileemd_15008_half_map_2.map
Projections & Slices
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Sample components

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Entire : Polyribosomes from HeLa cytoplasm

EntireName: Polyribosomes from HeLa cytoplasm
Components
  • Complex: Polyribosomes from HeLa cytoplasm

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Supramolecule #1: Polyribosomes from HeLa cytoplasm

SupramoleculeName: Polyribosomes from HeLa cytoplasm / type: complex / ID: 1 / Parent: 0
Details: Polysomes in cell lysate obtained by osmotic lysis of HeLa cells
Source (natural)Organism: Homo sapiens (human) / Strain: HeLa / Location in cell: cytoplasm

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferpH: 7.6
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 26.0 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsSpherical aberration corrector: Cs image corrector (CEOS, Germany)
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Average exposure time: 1.0 sec. / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 3783 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 3.5 µm / Nominal magnification: 18000
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

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 16.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number subtomograms used: 2976
ExtractionNumber tomograms: 36 / Number images used: 2976 / Reference model: de novo / Method: volumes picked interactively / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
FSC plot (resolution estimation)

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