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- EMDB-2597: Cryo-EM of a pretermination complex with eRF1 and eRF3 -

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Basic information

Entry
Database: EMDB / ID: EMD-2597
TitleCryo-EM of a pretermination complex with eRF1 and eRF3
Map dataComplex locked with nonhydrolyzable GTP analog GDPNP
Sample
  • Sample: "CMV"-stalled wheat germ 80S-RNC bound to eRF1 and eRF3-GDPNP
  • Complex: 80S ribosomeEukaryotic ribosome
  • Protein or peptide: Sup45
  • Protein or peptide: Sup35
Keywordstranslation / termination / cryo-EM
Function / homology
Function and homology information


Eukaryotic Translation Termination / cytoplasmic translational termination / translation release factor complex / translation release factor activity / translation release factor activity, codon specific / sequence-specific mRNA binding / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / aminoacyl-tRNA hydrolase activity / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) ...Eukaryotic Translation Termination / cytoplasmic translational termination / translation release factor complex / translation release factor activity / translation release factor activity, codon specific / sequence-specific mRNA binding / nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / aminoacyl-tRNA hydrolase activity / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / translational termination / DNA-templated transcription termination / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / cytoplasmic stress granule / translation / GTPase activity / mRNA binding / GTP binding / identical protein binding / cytosol / cytoplasm
Similarity search - Function
Eukaryotic peptide chain release factor GTP-binding subunit / 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 ...Eukaryotic peptide chain release factor GTP-binding subunit / 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 / Eukaryotic peptide chain release factor subunit 1
Similarity search - Component
Biological speciesTriticum aestivum (bread wheat) / Saccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.15 Å
AuthorsPreis A / Heuer A / Barrio-Garcia C / Hauser A / Eyler D / Berninghausen O / Green R / Becker T / Beckmann R
CitationJournal: Cell Rep / Year: 2014
Title: Cryoelectron microscopic structures of eukaryotic translation termination complexes containing eRF1-eRF3 or eRF1-ABCE1.
Authors: Anne Preis / Andre Heuer / Clara Barrio-Garcia / Andreas Hauser / Daniel E Eyler / Otto Berninghausen / Rachel Green / Thomas Becker / Roland Beckmann /
Abstract: Termination and ribosome recycling are essential processes in translation. In eukaryotes, a stop codon in the ribosomal A site is decoded by a ternary complex consisting of release factors eRF1 and ...Termination and ribosome recycling are essential processes in translation. In eukaryotes, a stop codon in the ribosomal A site is decoded by a ternary complex consisting of release factors eRF1 and guanosine triphosphate (GTP)-bound eRF3. After GTP hydrolysis, eRF3 dissociates, and ABCE1 can bind to eRF1-loaded ribosomes to stimulate peptide release and ribosomal subunit dissociation. Here, we present cryoelectron microscopic (cryo-EM) structures of a pretermination complex containing eRF1-eRF3 and a termination/prerecycling complex containing eRF1-ABCE1. eRF1 undergoes drastic conformational changes: its central domain harboring the catalytically important GGQ loop is either packed against eRF3 or swung toward the peptidyl transferase center when bound to ABCE1. Additionally, in complex with eRF3, the N-terminal domain of eRF1 positions the conserved NIKS motif proximal to the stop codon, supporting its suggested role in decoding, yet it appears to be delocalized in the presence of ABCE1. These results suggest that stop codon decoding and peptide release can be uncoupled during termination.
History
DepositionFeb 26, 2014-
Header (metadata) releaseMar 26, 2014-
Map releaseJul 16, 2014-
UpdateJul 23, 2014-
Current statusJul 23, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.183
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.183
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4crn
  • Surface level: 0.183
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4crn
  • Surface level: 0.183
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-4crn
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_2597.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationComplex locked with nonhydrolyzable GTP analog GDPNP
Voxel sizeX=Y=Z: 1.2375 Å
Density
Contour LevelBy AUTHOR: 0.183 / Movie #1: 0.183
Minimum - Maximum-0.455769 - 0.92238963
Average (Standard dev.)0.00892477 (±0.07670007)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-184-184-183
Dimensions368368368
Spacing368368368
CellA=B=C: 455.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.23751.23751.2375
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z455.400455.400455.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S213
start NC/NR/NS-184-184-183
NC/NR/NS368368368
D min/max/mean-0.4560.9220.009

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Supplemental data

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Sample components

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Entire : "CMV"-stalled wheat germ 80S-RNC bound to eRF1 and eRF3-GDPNP

EntireName: "CMV"-stalled wheat germ 80S-RNC bound to eRF1 and eRF3-GDPNP
Components
  • Sample: "CMV"-stalled wheat germ 80S-RNC bound to eRF1 and eRF3-GDPNP
  • Complex: 80S ribosomeEukaryotic ribosome
  • Protein or peptide: Sup45
  • Protein or peptide: Sup35

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Supramolecule #1000: "CMV"-stalled wheat germ 80S-RNC bound to eRF1 and eRF3-GDPNP

SupramoleculeName: "CMV"-stalled wheat germ 80S-RNC bound to eRF1 and eRF3-GDPNP
type: sample / ID: 1000
Oligomeric state: One ribosome binds to one eRF1 and one eRF3
Number unique components: 3

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Triticum aestivum (bread wheat) / synonym: Bread wheat / Tissue: seed

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Macromolecule #1: Sup45

MacromoleculeName: Sup45 / type: protein_or_peptide / ID: 1 / Name.synonym: eRF1 / Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 49 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pTYB2

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Macromolecule #2: Sup35

MacromoleculeName: Sup35 / type: protein_or_peptide / ID: 2 / Name.synonym: eRF3
Details: Sup35 was N-terminally deleted for the first 97 amino acids
Number of copies: 1 / Oligomeric state: 1 / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Location in cell: cytoplasm
Molecular weightTheoretical: 76 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pTYB2

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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 HEPES pH 7.5, 200 mM KCl, 1.5 MgCl2, 2 mM DTT, 0.01 mg/ml cycloheximide, 0.05 % Nikkol, 0.03 % DBC, 0.5 mM GDPNP).
StainingType: NEGATIVE / Details: Cryo-EM
GridDetails: Sec61 was added at a five-fold molar excess to saturate the hydrophobic signal-anchor sequence and avoid orientational bias on the cryo-grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 147136 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateFeb 20, 2013
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Digitization - Sampling interval: 15.6 µm / Average electron dose: 20 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: on volumes (SPIDER)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.15 Å / Resolution method: OTHER / Software - Name: Star, Fish, SPIDER / Details: Data-subset resulted from computational sorting / Number images used: 39309
DetailsThe particles were picked with starfish_boxing version 0.2.0, which is part of the new StarFish single particle analysis program suite.

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Atomic model buiding 1

Initial modelPDB ID:
SoftwareName: Chimera, Coot
DetailsA homology model for eRF1 was built using HHPred; The domains were separately fitted by manual docking using programs Coot and Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4crn:
Cryo-EM of a pretermination complex with eRF1 and eRF3

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Atomic model buiding 2

Initial modelPDB ID:
SoftwareName: Chimera, Coot
DetailsA homology model for eRF3 was built using HHPred; The domains were separately fitted by manual docking using programs Coot and Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-4crn:
Cryo-EM of a pretermination complex with eRF1 and eRF3

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