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- EMDB-2622: PRE-translocational (classical-2) 80S ribosomal state of Regulati... -

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Basic information

Entry
Database: EMDB / ID: EMD-2622
TitlePRE-translocational (classical-2) 80S ribosomal state of Regulation of the mammalian elongation cycle by 40S subunit rolling: a eukaryotic-specific ribosome rearrangement
Map datareconstruction of pre-translocational (classical-2) state of 80S ribosome
Sample
  • Sample: Pre-translocational (classical-2) rabbit 80S ribosome with three tRNAs and mRNA bound
  • Complex: Re-associated 80S
  • RNA: transfer RNA
  • RNA: transfer RNA
  • RNA: transfer RNA
  • RNA: messenger RNA
Keywordstranslation / mammalian 80S ribosome / elongation cycle / pre-translocational state / tRNA selection / cryo-electron microscopy
Biological speciesOryctolagus cuniculus (rabbit) / Bos taurus (cattle) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.5 Å
AuthorsBudkevich TV / Giesebrecht J / Behrmann E / Loerke J / Ramrath D / Mielke T / Ismer J / Hildebrand P / Tung C-S / Nierhaus KH ...Budkevich TV / Giesebrecht J / Behrmann E / Loerke J / Ramrath D / Mielke T / Ismer J / Hildebrand P / Tung C-S / Nierhaus KH / Sanbonmatsu KY / Spahn CMT
CitationJournal: Cell / Year: 2014
Title: Regulation of the mammalian elongation cycle by subunit rolling: a eukaryotic-specific ribosome rearrangement.
Authors: Tatyana V Budkevich / Jan Giesebrecht / Elmar Behrmann / Justus Loerke / David J F Ramrath / Thorsten Mielke / Jochen Ismer / Peter W Hildebrand / Chang-Shung Tung / Knud H Nierhaus / ...Authors: Tatyana V Budkevich / Jan Giesebrecht / Elmar Behrmann / Justus Loerke / David J F Ramrath / Thorsten Mielke / Jochen Ismer / Peter W Hildebrand / Chang-Shung Tung / Knud H Nierhaus / Karissa Y Sanbonmatsu / Christian M T Spahn /
Abstract: The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution ...The extent to which bacterial ribosomes and the significantly larger eukaryotic ribosomes share the same mechanisms of ribosomal elongation is unknown. Here, we present subnanometer resolution cryoelectron microscopy maps of the mammalian 80S ribosome in the posttranslocational state and in complex with the eukaryotic eEF1A⋅Val-tRNA⋅GMPPNP ternary complex, revealing significant differences in the elongation mechanism between bacteria and mammals. Surprisingly, and in contrast to bacterial ribosomes, a rotation of the small subunit around its long axis and orthogonal to the well-known intersubunit rotation distinguishes the posttranslocational state from the classical pretranslocational state ribosome. We term this motion "subunit rolling." Correspondingly, a mammalian decoding complex visualized in substates before and after codon recognition reveals structural distinctions from the bacterial system. These findings suggest how codon recognition leads to GTPase activation in the mammalian system and demonstrate that in mammalia subunit rolling occurs during tRNA selection.
History
DepositionMar 31, 2014-
Header (metadata) releaseApr 16, 2014-
Map releaseJul 9, 2014-
UpdateAug 20, 2014-
Current statusAug 20, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2622_500...

Downloads & links

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Map

FileDownload / File: emd_2622.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of pre-translocational (classical-2) state of 80S ribosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.26 Å/pix.
x 360 pix.
= 453.6 Å		1.26 Å/pix.
x 360 pix.
= 453.6 Å		1.26 Å/pix.
x 360 pix.
= 453.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.26 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-4.51747513 - 11.762286189999999
Average (Standard dev.)0.2124048 (±0.94575697)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin180180180
Dimensions360360360
Spacing360360360
CellA=B=C: 453.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.261.261.26
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z453.600453.600453.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S123
start NC/NR/NS180180180
NC/NR/NS360360360
D min/max/mean-4.51711.7620.212

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Supplemental data

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Sample components

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Entire : Pre-translocational (classical-2) rabbit 80S ribosome with three ...

EntireName: Pre-translocational (classical-2) rabbit 80S ribosome with three tRNAs and mRNA bound
Components
  • Sample: Pre-translocational (classical-2) rabbit 80S ribosome with three tRNAs and mRNA bound
  • Complex: Re-associated 80S
  • RNA: transfer RNA
  • RNA: transfer RNA
  • RNA: transfer RNA
  • RNA: messenger RNA

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Supramolecule #1000: Pre-translocational (classical-2) rabbit 80S ribosome with three ...

SupramoleculeName: Pre-translocational (classical-2) rabbit 80S ribosome with three tRNAs and mRNA bound
type: sample / ID: 1000
Details: 80S were re-associated from 40S and 60S subunits and the sample assembled in vitro from individual components
Oligomeric state: one 80S binds three tRNAs and one mRNA / Number unique components: 5
Molecular weightTheoretical: 4.5 MDa

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Supramolecule #1: Re-associated 80S

SupramoleculeName: Re-associated 80S / type: complex / ID: 1 / Name.synonym: 80S ribosome / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit / Tissue: Liver
Molecular weightTheoretical: 4.5 MDa

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Macromolecule #1: transfer RNA

MacromoleculeName: transfer RNA / type: rna / ID: 1 / Name.synonym: tRNA / Details: tRNAPhe / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit
Molecular weightTheoretical: 25 KDa

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Macromolecule #2: transfer RNA

MacromoleculeName: transfer RNA / type: rna / ID: 2 / Name.synonym: tRNA / Details: tRNALys, isoacceptor 3 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Bos taurus (cattle) / synonym: Bovine
Molecular weightTheoretical: 25 KDa

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Macromolecule #3: transfer RNA

MacromoleculeName: transfer RNA / type: rna / ID: 3 / Name.synonym: tRNA / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit
Molecular weightTheoretical: 25 KDa

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Macromolecule #4: messenger RNA

MacromoleculeName: messenger RNA / type: rna / ID: 4 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: XL1-blue / synonym: bacteria
Molecular weightTheoretical: 14.1 KDa
SequenceString:
GGGAAAAGAA AAGAAAAGAA AAUGUUCAAA GAAAAGAAAA GAAAU

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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

Concentration1.38 mg/mL
BufferpH: 7.5
Details: 20 mM Hepes, 5mM MgCl2, 100 mM NH4Cl, 6 mM beta-mercaptoethanol, 0.8 mM spermidine, 0.6 mM spermine
GridDetails: Quantifoil grids with additional continuous carbon support.
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Instrument: FEI VITROBOT MARK II / Method: blot for 2/4 sec before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 77 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification
Detailsminimal dose system
DateJun 2, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Number real images: 826 / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 65520 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 39000
Sample stageSpecimen holder: Nitrogen cooled / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using SIGNATURE and processed by using SPIDER and Sparx
CTF correctionDetails: defocus group
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.5 Å / Resolution method: OTHER / Software - Name: Spider, Sparx / Number images used: 73951

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