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Entry
Database: EMDB / ID: EMD-5801
TitleStructure of the mammalian ribosomal pre-termination complex associated with eRF1-eRF3-GDPNP
Map dataReconstruction of the eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex assembled in vitro on a derivative of beta-globin mRNA encoding MVHL tetrapeptide followed by a UAA stop codon. The P-site is occupied by a tRNAleu.
Sample
  • Sample: eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex
  • Complex: 80S ribosomeEukaryotic ribosome
  • Protein or peptide: eukaryotic release factor 1
  • Protein or peptide: eukaryotic release factor 3
  • RNA: Transfer-messenger RNA Leu
  • RNA: messenger RNA
KeywordsTranslation termination / eRF1 / eRF3 / tRNAleu / ribosome / mammalian
Function / homology
Function and homology information


translation termination factor activity / cytoplasmic translational termination / translation release factor complex / regulation of translational termination / translation release factor activity / translation release factor activity, codon specific / protein methylation / sequence-specific mRNA binding / aminoacyl-tRNA hydrolase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay ...translation termination factor activity / cytoplasmic translational termination / translation release factor complex / regulation of translational termination / translation release factor activity / translation release factor activity, codon specific / protein methylation / sequence-specific mRNA binding / aminoacyl-tRNA hydrolase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / Protein hydroxylation / Eukaryotic Translation Termination / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / translational termination / cytosolic ribosome / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / G1/S transition of mitotic cell cycle / Regulation of expression of SLITs and ROBOs / ribosome binding / translation / GTPase activity / GTP binding / RNA binding / cytosol / cytoplasm
Similarity search - Function
Peptide chain release factor eRF1/aRF1 / eRF1, domain 1 / eRF1 domain 2 / eRF1 domain 2 / eRF1 domain 1 / eRF1 domain 1/Pelota-like / eRF1 domain 3 / eRF1, domain 2 superfamily / eRF1 domain 3 / eRF1_1 ...Peptide chain release factor eRF1/aRF1 / eRF1, domain 1 / eRF1 domain 2 / eRF1 domain 2 / eRF1 domain 1 / eRF1 domain 1/Pelota-like / eRF1 domain 3 / eRF1, domain 2 superfamily / eRF1 domain 3 / eRF1_1 / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / 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 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / 50S ribosomal protein L30e-like / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Eukaryotic peptide chain release factor GTP-binding subunit ERF3A / Eukaryotic peptide chain release factor subunit 1
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.7 Å
Authorsdes Georges A / Hashem H / Unbehaun A / Grassucci RA / Taylor D / Hellen CUT / Pestova TV / Frank J
CitationJournal: Nucleic Acids Res / Year: 2014
Title: Structure of the mammalian ribosomal pre-termination complex associated with eRF1.eRF3.GDPNP.
Authors: Amédée des Georges / Yaser Hashem / Anett Unbehaun / Robert A Grassucci / Derek Taylor / Christopher U T Hellen / Tatyana V Pestova / Joachim Frank /
Abstract: Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl- ...Eukaryotic translation termination results from the complex functional interplay between two release factors, eRF1 and eRF3, in which GTP hydrolysis by eRF3 couples codon recognition with peptidyl-tRNA hydrolysis by eRF1. Here, we present a cryo-electron microscopy structure of pre-termination complexes associated with eRF1•eRF3•GDPNP at 9.7 -Å resolution, which corresponds to the initial pre-GTP hydrolysis stage of factor attachment and stop codon recognition. It reveals the ribosomal positions of eRFs and provides insights into the mechanisms of stop codon recognition and triggering of eRF3's GTPase activity.
History
SupersessionID: EMD-5518
DepositionNov 21, 2013-
Header (metadata) releaseDec 25, 2013-
Map releaseDec 25, 2013-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j5y
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j5y
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5801.jpg

Downloads & links

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Map

FileDownload / File: emd_5801.map.gz / Format: CCP4 / Size: 104.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex assembled in vitro on a derivative of beta-globin mRNA encoding MVHL tetrapeptide followed by a UAA stop codon. The P-site is occupied by a tRNAleu.
Voxel sizeX=Y=Z: 1.5 Å
Density
Contour LevelBy AUTHOR: 0.02 / Movie #1: 0.02
Minimum - Maximum-0.02488585 - 0.08083032
Average (Standard dev.)0.00140791 (±0.0087062)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions304304304
Spacing304304304
CellA=B=C: 456.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.51.51.5
M x/y/z304304304
origin x/y/z0.0000.0000.000
length x/y/z456.000456.000456.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS304304304
D min/max/mean-0.0250.0810.001

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Supplemental data

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Sample components

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Entire : eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex

EntireName: eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex
Components
  • Sample: eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex
  • Complex: 80S ribosomeEukaryotic ribosome
  • Protein or peptide: eukaryotic release factor 1
  • Protein or peptide: eukaryotic release factor 3
  • RNA: Transfer-messenger RNA Leu
  • RNA: messenger RNA

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Supramolecule #1000: eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex

SupramoleculeName: eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complex
type: sample / ID: 1000 / Number unique components: 5
Molecular weightTheoretical: 4.5 MDa / Method: calculation

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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: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Tissue: blood / Cell: red blood cells
Molecular weightTheoretical: 4.5 MDa

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Macromolecule #1: eukaryotic release factor 1

MacromoleculeName: eukaryotic release factor 1 / type: protein_or_peptide / ID: 1 / Name.synonym: eRF1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightTheoretical: 480 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Eukaryotic peptide chain release factor subunit 1

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Macromolecule #2: eukaryotic release factor 3

MacromoleculeName: eukaryotic release factor 3 / type: protein_or_peptide / ID: 2 / Name.synonym: eRF3 / Details: 138 aa N-terminal deletion / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: human
Molecular weightTheoretical: 540 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Eukaryotic peptide chain release factor GTP-binding subunit ERF3A

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Macromolecule #3: Transfer-messenger RNA Leu

MacromoleculeName: Transfer-messenger RNA Leu / type: rna / ID: 3 / Name.synonym: tRNAleu
Details: Native tRNAs were aminoacylated with Met, Val, His, and Leu using native aminoacyl-tRNA synthetases.
Classification: OTHER / Structure: OTHER / 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
Details: beta-globin mRNA encoding MVHL tetrapeptide followed by a UAA stop codon
Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: 20 mM Tris, 100 mM KOAc, 2.5 mM MgCl2, 2 mM DTT, 0.25 mM spermidine supplemented with 200 units RNasin, 0.4 mM ATP, 3 mM Mg-GMPPNP
StainingType: NEGATIVE / Details: cryo
GridDetails: Quantifoil grids, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm
Sample stageSpecimen holder model: GATAN HELIUM
DateJan 1, 2010
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k) / Average electron dose: 25 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each Particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.7 Å / Resolution method: OTHER / Software - Name: RELION / Number images used: 48973
DetailsImages were classified and refined with RELION.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3e1y


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: F
SoftwareName: Chimera
DetailsDomains were fitted as rigid bodies using Chimera. Individual domains were then refined manually using hinge points as points of flexibility.
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross correlation
Output model

PDB-3j5y:
Structure of the mammalian ribosomal pre-termination complex associated with eRF1-eRF3-GDPNP

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Atomic model buiding 2

Initial modelPDB ID:

2ktu


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

PDB-3j5y:
Structure of the mammalian ribosomal pre-termination complex associated with eRF1-eRF3-GDPNP

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Atomic model buiding 3

Initial modelPDB ID:

1r5b


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

PDB-3j5y:
Structure of the mammalian ribosomal pre-termination complex associated with eRF1-eRF3-GDPNP

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Atomic model buiding 4

Initial modelPDB ID:

1r5o


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

PDB-3j5y:
Structure of the mammalian ribosomal pre-termination complex associated with eRF1-eRF3-GDPNP

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Atomic model buiding 5

Initial modelPDB ID:

3vmf


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

PDB-3j5y:
Structure of the mammalian ribosomal pre-termination complex associated with eRF1-eRF3-GDPNP

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