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Entry
Database: EMDB / ID: EMD-5977
TitleStructures of yeast 80S ribosome-tRNA complexes in the rotated and non-rotated conformations (Class I - 2 tRNA in non-rotated conformation)
Map dataReconstruction of a yeast 80S ribosome in the classical state with 2 tRNA bound. (Class I)
Sample
  • Sample: 80S ribosome bound to mRNA containing Kozak sequence and to two tRNA
  • Complex: 80S ribosome
  • RNA: transfer RNA
  • RNA: mRNA
Keywords80S ribosome / Kozak sequence / translation
Function / homology
Function and homology information


maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, LSU-rRNA,5S) / Negative regulators of DDX58/IFIH1 signaling / regulation of amino acid metabolic process / negative regulation of glucose mediated signaling pathway / positive regulation of translational fidelity / RMTs methylate histone arginines / Protein methylation / mTORC1-mediated signalling / Protein hydroxylation / ribosome-associated ubiquitin-dependent protein catabolic process ...maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, LSU-rRNA,5S) / Negative regulators of DDX58/IFIH1 signaling / regulation of amino acid metabolic process / negative regulation of glucose mediated signaling pathway / positive regulation of translational fidelity / RMTs methylate histone arginines / Protein methylation / mTORC1-mediated signalling / Protein hydroxylation / ribosome-associated ubiquitin-dependent protein catabolic process / GDP-dissociation inhibitor activity / positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / pre-mRNA 5'-splice site binding / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition / preribosome, small subunit precursor / nonfunctional rRNA decay / response to cycloheximide / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Major pathway of rRNA processing in the nucleolus and cytosol / mRNA destabilization / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / negative regulation of translational frameshifting / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / negative regulation of mRNA splicing, via spliceosome / Formation of a pool of free 40S subunits / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translational elongation / ribosomal large subunit export from nucleus / G-protein alpha-subunit binding / 90S preribosome / positive regulation of protein kinase activity / Ub-specific processing proteases / ribosomal subunit export from nucleus / regulation of translational fidelity / protein-RNA complex assembly / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translational termination / maturation of LSU-rRNA / ribosomal small subunit export from nucleus / translation regulator activity / DNA-(apurinic or apyrimidinic site) endonuclease activity / rescue of stalled ribosome / cellular response to amino acid starvation / ribosome assembly / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ribosomal large subunit biogenesis / protein kinase C binding / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / translational initiation / small-subunit processome / macroautophagy / maintenance of translational fidelity / modification-dependent protein catabolic process / protein tag activity / cytoplasmic stress granule / rRNA processing / ribosome biogenesis / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / small ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / negative regulation of translation / rRNA binding / protein ubiquitination / ribosome / structural constituent of ribosome / G protein-coupled receptor signaling pathway / translation / negative regulation of gene expression / response to antibiotic / mRNA binding / ubiquitin protein ligase binding / nucleolus / mitochondrion / RNA binding / zinc ion binding / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / 50S ribosomal protein L10, insertion domain superfamily / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / : / : / Ribosomal protein S26e signature. / Ribosomal protein L41 ...60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / : / 50S ribosomal protein L10, insertion domain superfamily / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / : / : / Ribosomal protein S26e signature. / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / Ribosomal protein L1, conserved site / Ribosomal protein S26e / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein L1 signature. / : / Ribosomal protein S12e signature. / Ribosomal protein L1 / Ribosomal protein S12e / Ribosomal protein L29e / Ribosomal L29e protein family / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Small (40S) ribosomal subunit Asc1/RACK1 / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal protein S2, eukaryotic / S27a-like superfamily / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein L10e, conserved site / Ribosomal protein L10e signature. / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein L10e / 40S Ribosomal protein S10 / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal L38e protein family / : / Ribosomal protein S7e signature. / Ribosomal protein L44e signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal protein L24e, conserved site / Ribosomal protein L24e signature. / : / Ribosomal protein S10, eukaryotic/archaeal / Ribosomal protein L19, eukaryotic / Ribosomal protein L19/L19e conserved site / Ribosomal protein L19e signature. / Ribosomal protein L6e signature. / Ribosomal protein L13e / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein L13e / 60S ribosomal protein L18a/ L20, eukaryotes / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein S27a / Ribosomal protein S17e, conserved site / : / Ribosomal protein S27a / Ribosomal protein S17e signature. / Ribosomal protein S27a / Ribosomal protein S3Ae, conserved site / Ribosomal protein S3Ae signature. / Ribosomal protein L44e / Ribosomal protein S30 / Ribosomal protein L44 / Ribosomal protein S30 / Ribosomal protein S2, eukaryotic/archaeal / : / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein L30e signature 1. / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Ribosomal L40e family / Ribosomal protein S27e signature. / Ribosomal protein 50S-L18Ae/60S-L20/60S-L18A / 40S ribosomal protein S4, C-terminal domain / Ribosomal proteins 50S-L18Ae/60S-L20/60S-L18A / 40S ribosomal protein S4 C-terminus / Ribosomal protein S4e, N-terminal, conserved site / Ribosomal protein S4e signature. / Ribosomal protein L23/L25, N-terminal / Ribosomal protein L23, N-terminal domain / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal_L40e
Similarity search - Domain/homology
Small ribosomal subunit protein uS4A / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein eS17A / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A ...Small ribosomal subunit protein uS4A / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein eS17A / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL29 / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL22A / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS15 / Ubiquitin-ribosomal protein eS31 fusion protein / Small ribosomal subunit protein uS11A / Small ribosomal subunit protein eS19A / Small ribosomal subunit protein eS21A / Small ribosomal subunit protein uS8A / Large ribosomal subunit protein uL5A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Ubiquitin-ribosomal protein eL40A fusion protein / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein eL42A / Small ribosomal subunit protein uS12A / Small ribosomal subunit protein eS24A / Small ribosomal subunit protein eS30A / Small ribosomal subunit protein eS4A / Small ribosomal subunit protein eS6A / Small ribosomal subunit protein eS8A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL1A / Large ribosomal subunit protein uL2A / Small ribosomal subunit protein uS17A / Large ribosomal subunit protein eL18A / Small ribosomal subunit protein uS9A / Large ribosomal subunit protein uL11A / Small ribosomal subunit protein uS13A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Small ribosomal subunit protein eS32A / Large ribosomal subunit protein uL4A / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein uL18 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL13A / Small ribosomal subunit protein eS7A / Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS1A / Small ribosomal subunit protein eS27A / Large ribosomal subunit protein eL14A / Small ribosomal subunit protein RACK1 / Large ribosomal subunit protein eL32 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein eS26A / Small ribosomal subunit protein uS14A / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein eS12 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Large ribosomal subunit protein eL34A / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Small ribosomal subunit protein eS10A / Large ribosomal subunit protein eL13A / Small ribosomal subunit protein eS25A / Small ribosomal subunit protein eS28A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.3 Å
AuthorsSvidritskiy E / Brilot AF / Koh CS / Grigorieff N / Korostelev AA
CitationJournal: Structure / Year: 2014
Title: Structures of yeast 80S ribosome-tRNA complexes in the rotated and nonrotated conformations.
Authors: Egor Svidritskiy / Axel F Brilot / Cha San Koh / Nikolaus Grigorieff / Andrei A Korostelev /
Abstract: The structural understanding of eukaryotic translation lags behind that of translation on bacterial ribosomes. Here, we present two subnanometer resolution structures of S. cerevisiae 80S ribosome ...The structural understanding of eukaryotic translation lags behind that of translation on bacterial ribosomes. Here, we present two subnanometer resolution structures of S. cerevisiae 80S ribosome complexes formed with either one or two tRNAs and bound in response to an mRNA fragment containing the Kozak consensus sequence. The ribosomes adopt two globally different conformations that are related to each other by the rotation of the small subunit. Comparison with bacterial ribosome complexes reveals that the global structures and modes of intersubunit rotation of the yeast ribosome differ significantly from those in the bacterial counterpart, most notably in the regions involving the tRNA, small ribosomal subunit, and conserved helix 69 of the large ribosomal subunit. The structures provide insight into ribosome dynamics implicated in tRNA translocation and help elucidate the role of the Kozak fragment in positioning an open reading frame during translation initiation in eukaryotes.
History
DepositionMay 21, 2014-
Header (metadata) releaseJul 30, 2014-
Map releaseAug 6, 2014-
UpdateOct 7, 2015-
Current statusOct 7, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j78
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_5977.gif

Downloads & links

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Map

FileDownload / File: emd_5977.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a yeast 80S ribosome in the classical state with 2 tRNA bound. (Class I)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 360 pix.
= 378. Å		1.05 Å/pix.
x 360 pix.
= 378. Å		1.05 Å/pix.
x 360 pix.
= 378. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.05 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.19798017 - 0.42953041
Average (Standard dev.)0.00601077 (±0.04799228)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 377.99997 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.051.051.05
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-800-4
NX/NY/NZ1611358
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-0.1980.4300.006

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Supplemental data

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Sample components

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Entire : 80S ribosome bound to mRNA containing Kozak sequence and to two tRNA

EntireName: 80S ribosome bound to mRNA containing Kozak sequence and to two tRNA
Components
  • Sample: 80S ribosome bound to mRNA containing Kozak sequence and to two tRNA
  • Complex: 80S ribosome
  • RNA: transfer RNA
  • RNA: mRNA

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Supramolecule #1000: 80S ribosome bound to mRNA containing Kozak sequence and to two tRNA

SupramoleculeName: 80S ribosome bound to mRNA containing Kozak sequence and to two tRNA
type: sample / ID: 1000 / Details: Sample was monodisperse. / Number unique components: 3
Molecular weightExperimental: 3.5 MDa

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Yeast
Molecular weightExperimental: 3.5 MDa

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Macromolecule #1: transfer RNA

MacromoleculeName: transfer RNA / type: rna / ID: 1 / Name.synonym: tRNA / Details: tRNA fmet / Classification: TRANSFER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25 KDa

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Macromolecule #2: mRNA

MacromoleculeName: mRNA / type: rna / ID: 2 / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 5 KDa
SequenceString:
AAAAAUGUAA AAAA

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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

Concentration1.2 mg/mL
BufferpH: 7.5
Details: 20 mM Tris-HCl, 50 mM NH4Cl, 20 mM MgCl2, 0.3 U/uL RNasin
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateJan 2, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 4754 / Average electron dose: 30 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 133333 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 4.844 µm / Nominal defocus min: 1.159 µm / Nominal magnification: 133333
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND3, FREALIGN per micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.3 Å / Resolution method: OTHER / Software - Name: EMAN2, IMAGIC, FREALIGN, RSAMPLE, CTFFIND3 / Number images used: 23163

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Atomic model buiding 1

Initial modelPDB ID:

3u5b
PDB Unreleased entry

SoftwareName: Chimera, CNS
Details3U5B, 3U5C, 3U5D, and 3U5E were combined prior to fitting. tRNAs and mRNA were modeled using individual tRNAs and mRNA from the crystal structure (3I9B) of the classical-state 70S ribosome. The structure of rpL1 was obtained by homology modeling from PDB ID 3J3B.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j78:
Structures of yeast 80S ribosome-tRNA complexes in the rotated and non-rotated conformations (Class I - non-rotated ribosome with 2 tRNAs)

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Atomic model buiding 2

Initial modelPDB ID:

3u5c
PDB Unreleased entry

SoftwareName: Chimera, CNS
Details3U5B, 3U5C, 3U5D, and 3U5E were combined prior to fitting. tRNAs and mRNA were modeled using individual tRNAs and mRNA from the crystal structure (3I9B) of the classical-state 70S ribosome. The structure of rpL1 was obtained by homology modeling from PDB ID 3J3B.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j78:
Structures of yeast 80S ribosome-tRNA complexes in the rotated and non-rotated conformations (Class I - non-rotated ribosome with 2 tRNAs)

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Atomic model buiding 3

Initial modelPDB ID:

3u5d
PDB Unreleased entry

SoftwareName: Chimera, CNS
Details3U5B, 3U5C, 3U5D, and 3U5E were combined prior to fitting. tRNAs and mRNA were modeled using individual tRNAs and mRNA from the crystal structure (3I9B) of the classical-state 70S ribosome. The structure of rpL1 was obtained by homology modeling from PDB ID 3J3B.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j78:
Structures of yeast 80S ribosome-tRNA complexes in the rotated and non-rotated conformations (Class I - non-rotated ribosome with 2 tRNAs)

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Atomic model buiding 4

Initial modelPDB ID:

3u5e
PDB Unreleased entry

SoftwareName: Chimera, CNS
Details3U5B, 3U5C, 3U5D, and 3U5E were combined prior to fitting. tRNAs and mRNA were modeled using individual tRNAs and mRNA from the crystal structure (3I9B) of the classical-state 70S ribosome. The structure of rpL1 was obtained by homology modeling from PDB ID 3J3B.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j78:
Structures of yeast 80S ribosome-tRNA complexes in the rotated and non-rotated conformations (Class I - non-rotated ribosome with 2 tRNAs)

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Atomic model buiding 5

Initial modelPDB ID:

3i9b
PDB Unreleased entry


Chain - #0 - Chain ID: 1 / Chain - #1 - Chain ID: C / Chain - #2 - Chain ID: D
SoftwareName: Chimera, CNS
Details3U5B, 3U5C, 3U5D, and 3U5E were combined prior to fitting. tRNAs and mRNA were modeled using individual tRNAs and mRNA from the crystal structure (3I9B) of the classical-state 70S ribosome. The structure of rpL1 was obtained by homology modeling from PDB ID 3J3B.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j78:
Structures of yeast 80S ribosome-tRNA complexes in the rotated and non-rotated conformations (Class I - non-rotated ribosome with 2 tRNAs)

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Atomic model buiding 6

Initial modelPDB ID:

3j3b
PDB Unreleased entry

SoftwareName: Chimera, CNS
Details3U5B, 3U5C, 3U5D, and 3U5E were combined prior to fitting. tRNAs and mRNA were modeled using individual tRNAs and mRNA from the crystal structure (3I9B) of the classical-state 70S ribosome. The structure of rpL1 was obtained by homology modeling from PDB ID 3J3B.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: cross-correlation
Output model

PDB-3j78:
Structures of yeast 80S ribosome-tRNA complexes in the rotated and non-rotated conformations (Class I - non-rotated ribosome with 2 tRNAs)

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