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- EMDB-2623: Sampling intermediate of Regulation of the mammalian elongation c... -

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Basic information

Entry
Database: EMDB / ID: EMD-2623
TitleSampling intermediate of Regulation of the mammalian elongation cycle by 40S subunit rolling: a eukaryotic-specific ribosome rearrangement
Map datareconstruction of initial codon-sampling state of 80S
Sample
  • Sample: Initial codon sampling state of 80S mammalian ribosome
  • Complex: Re-associated 80S
  • RNA: transfer RNA
  • RNA: transfer RNA
  • RNA: transfer RNA
  • RNA: messenger RNA
  • Protein or peptide: elongation factor 1A
Keywordstranslation / mammalian 80S ribosome / elongation cycle / tRNA selection / eukaryotic ternary complex / elongation factor eEF1A / cryo-electron microscopy
Function / homology
Function and homology information


guanyl nucleotide binding / kinase activator activity / protein-synthesizing GTPase / Formation of the ternary complex, and subsequently, the 43S complex / Ribosomal scanning and start codon recognition / Translation initiation complex formation / Protein hydroxylation / SARS-CoV-1 modulates host translation machinery / Peptide chain elongation / Selenocysteine synthesis ...guanyl nucleotide binding / kinase activator activity / protein-synthesizing GTPase / Formation of the ternary complex, and subsequently, the 43S complex / Ribosomal scanning and start codon recognition / Translation initiation complex formation / Protein hydroxylation / SARS-CoV-1 modulates host translation machinery / Peptide chain elongation / Selenocysteine synthesis / Formation of a pool of free 40S subunits / Eukaryotic Translation Termination / Response of EIF2AK4 (GCN2) to amino acid deficiency / SRP-dependent cotranslational protein targeting to membrane / Viral mRNA Translation / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / GTP hydrolysis and joining of the 60S ribosomal subunit / L13a-mediated translational silencing of Ceruloplasmin expression / translation elongation factor activity / Major pathway of rRNA processing in the nucleolus and cytosol / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / rough endoplasmic reticulum / cytosolic ribosome / stress granule assembly / cellular response to epidermal growth factor stimulus / small-subunit processome / Regulation of expression of SLITs and ROBOs / maintenance of translational fidelity / ribosomal small subunit biogenesis / cytosolic small ribosomal subunit / SARS-CoV-2 modulates host translation machinery / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / cytoplasmic translation / structural constituent of ribosome / ribosome / translation / GTPase activity / synapse / GTP binding / nucleolus / endoplasmic reticulum / RNA binding / nucleoplasm / nucleus / membrane / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Translation elongation factor EF1A, eukaryotic/archaeal / : / GTP-eEF1A C-terminal domain-like / : / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Ribosomal protein S23, eukaryotic/archaeal ...Translation elongation factor EF1A, eukaryotic/archaeal / : / GTP-eEF1A C-terminal domain-like / : / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Ribosomal protein S23, eukaryotic/archaeal / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Ribosomal protein S12 signature. / Ribosomal protein S12/S23 / Ribosomal protein S12/S23 / Translation protein, beta-barrel domain superfamily / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Small ribosomal subunit protein uS12 / Elongation factor 1-alpha 1 / Elongation factor 1-alpha
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Escherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.7 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(CAMBRIDGE,MASS.) / Year: 2014
Title: Regulation of the mammalian elongation cycle by 40S subunit rolling: a eukaryotic-specific ribosome rearrangement
Authors: 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
History
DepositionApr 2, 2014-
Header (metadata) releaseApr 16, 2014-
Map releaseJul 9, 2014-
UpdateJul 16, 2014-
Current statusJul 16, 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
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  • Surface view colored by height
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4cxg
  • Surface level: 3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4cxg
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol

Downloads & links

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Map

FileDownload / File: emd_2623.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of initial codon-sampling state of 80S
Voxel sizeX=Y=Z: 1.26 Å
Density
Contour LevelBy AUTHOR: 3.0 / Movie #1: 3
Minimum - Maximum-5.35204649 - 12.310511590000001
Average (Standard dev.)0.23774731 (±0.9626497)
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-5.35212.3110.238

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Supplemental data

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Sample components

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Entire : Initial codon sampling state of 80S mammalian ribosome

EntireName: Initial codon sampling state of 80S mammalian ribosome
Components
  • Sample: Initial codon sampling state of 80S mammalian ribosome
  • Complex: Re-associated 80S
  • RNA: transfer RNA
  • RNA: transfer RNA
  • RNA: transfer RNA
  • RNA: messenger RNA
  • Protein or peptide: elongation factor 1A

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Supramolecule #1000: Initial codon sampling state of 80S mammalian ribosome

SupramoleculeName: Initial codon sampling state of 80S mammalian ribosome
type: sample / ID: 1000
Details: 80S were re-associated from 40S and 60S subunits and the sample was assembled in vitro from individual components
Oligomeric state: one 80S binds three tRNAs, one elongation factor eEF1A and one mRNA
Number unique components: 6
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 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit
Molecular weightTheoretical: 25 KDa

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Macromolecule #3: transfer RNA

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

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Macromolecule #5: messenger RNA

MacromoleculeName: messenger RNA / type: rna / ID: 5 / Name.synonym: mRNA / Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Strain: XL-1 blue
Molecular weightTheoretical: 15 KDa
SequenceString:
GGGAAAAGAA AAGAAAAGAA AAUGUUCGUU AAAGAAAAGA AAAGAAAU

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Macromolecule #4: elongation factor 1A

MacromoleculeName: elongation factor 1A / type: protein_or_peptide / ID: 4 / Name.synonym: eEF1A / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit
Molecular weightTheoretical: 50 KDa
SequenceUniProtKB: Elongation factor 1-alpha 1

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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

Concentration1.3 mg/mL
BufferpH: 7.5
Details: 20 mM Hepes, 5 mM 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 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
DateSep 22, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Number real images: 230 / 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: 8.7 Å / Resolution method: OTHER / Software - Name: Spider, Sparx / Number images used: 79705

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Atomic model buiding 1

Initial modelPDB ID:

3vmf


Chain - Chain ID: A
SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation
Output model

PDB-4cxg:
Regulation of the mammalian elongation cycle by 40S subunit rolling: a eukaryotic-specific ribosome rearrangement

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Atomic model buiding 2

Initial modelPDB ID:

2xqd


Chain - Chain ID: Y
SoftwareName: Chimera, Coot
DetailsAcceptor stem loop (2XQD) and tRNA body (1TTT)(cutting points 26-27, 43-44) were docked separately as rigid bodies. Cutting points were restored using Coot.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation
Output model

PDB-4cxg:
Regulation of the mammalian elongation cycle by 40S subunit rolling: a eukaryotic-specific ribosome rearrangement

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Atomic model buiding 3

Initial modelPDB ID:

1ttt


Chain - Chain ID: D
SoftwareName: Chimera, Coot
DetailsAcceptor stem loop (2XQD) and tRNA body (1TTT)(cutting points 26-27, 43-44) were docked separately as rigid bodies. Cutting points were restored using Coot.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation
Output model

PDB-4cxg:
Regulation of the mammalian elongation cycle by 40S subunit rolling: a eukaryotic-specific ribosome rearrangement

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