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- PDB-3j5y: Structure of the mammalian ribosomal pre-termination complex asso... -

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Basic information

Entry
Database: PDB / ID: 3j5y
TitleStructure of the mammalian ribosomal pre-termination complex associated with eRF1-eRF3-GDPNP
Components
  • 5'-R(*AP*UP*UP*GP*UP*AP*AP*AP*AP*A)-3'
  • Eukaryotic peptide chain release factor GTP-binding subunit ERF3A
  • Eukaryotic peptide chain release factor subunit 1
  • tRNA-Leu
KeywordsTRANSLATION/RNA / Translation termination / eRF1 / eRF3 / tRNAleu / ribosome / mammalian / TRANSLATION-RNA complex
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
RNA / RNA (> 10) / Eukaryotic peptide chain release factor GTP-binding subunit ERF3A / Eukaryotic peptide chain release factor subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Oryctolagus cuniculus (rabbit)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 9.7 Å
Authorsdes Georges, A. / Hashem, Y. / Unbehaun, A. / Grassucci, R.A. / Taylor, D. / Hellen, C.U.T. / Pestova, T.V. / 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
DepositionNov 21, 2013Deposition site: RCSB / Processing site: RCSB
SupersessionDec 25, 2013ID: 3J2K
Revision 1.0Dec 25, 2013Provider: repository / Type: Initial release
Revision 1.1Mar 26, 2014Group: Database references
Revision 1.2Jul 18, 2018Group: Data collection / Category: em_image_scans / em_software / Item: _em_software.image_processing_id / _em_software.name

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Structure visualization

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Assembly

Deposited unit
A: Eukaryotic peptide chain release factor subunit 1
B: Eukaryotic peptide chain release factor GTP-binding subunit ERF3A
C: 5'-R(*AP*UP*UP*GP*UP*AP*AP*AP*AP*A)-3'
D: tRNA-Leu


Theoretical massNumber of molelcules
Total (without water)126,0794
Polymers126,0794
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein Eukaryotic peptide chain release factor subunit 1 / Eukaryotic release factor 1 / eRF1 / Protein Cl1 / TB3-1


Mass: 46478.121 Da / Num. of mol.: 1 / Fragment: UNP residues 7-420
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ETF1, ERF1, RF1, SUP45L1 / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: P62495
#2: Protein Eukaryotic peptide chain release factor GTP-binding subunit ERF3A / Eukaryotic peptide chain release factor subunit 3a / eRF3a / G1 to S phase transition protein 1 homolog


Mass: 48030.508 Da / Num. of mol.: 1 / Fragment: UNP residues 69-496
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: GSPT1, ERF3A / Production host: Escherichia coli (E. coli) / Strain (production host): BL21 / References: UniProt: P15170
#3: RNA chain 5'-R(*AP*UP*UP*GP*UP*AP*AP*AP*AP*A)-3'


Mass: 3193.980 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: messenger RNA / Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli)
#4: RNA chain tRNA-Leu


Mass: 28376.889 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Oryctolagus cuniculus (rabbit)

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeParent-ID
1eRF1-eRF3-GDPNP-bound mammalian ribosomal pre-termination complexRIBOSOME0
280S ribosomeEukaryotic ribosomeRIBOSOME1
3eukaryotic release factor 11
4eukaryotic release factor 31
5Transfer-messenger RNA Leu1
6messenger RNA1
Molecular weightValue: 4.5 MDa / Experimental value: NO
Buffer solutionName: 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
pH: 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
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Quantifoil grids, glow discharged
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temp: 93 K / Humidity: 100 % / Details: plunged into liquid ethane (FEI VITROBOT MARK IV)

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Jan 1, 2010
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Cs: 2.1 mm
Specimen holderSpecimen holder model: GATAN HELIUM / Temperature: 81 K
Image recordingElectron dose: 25 e/Å2 / Film or detector model: TVIPS TEMCAM-F415 (4k x 4k)
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameCategory
1UCSF Chimeramodel fitting
2RELION3D reconstruction
CTF correctionDetails: Each Particle
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionMethod: reference-based reconstruction / Resolution: 9.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 48973
Details: (Single particle details: Images were classified and refined with RELION) (Single particle--Applied symmetry: C1)
Symmetry type: POINT
Atomic model building
IDProtocolSpaceTarget criteriaDetails
1RIGID BODY FITREALCross correlationREFINEMENT PROTOCOL--RIGID BODY DETAILS--Domains were fitted as rigid bodies using Chimera. Individual domains were then refined manually using hinge points as points of flexibility.
2RIGID BODY FITREALCross correlationREFINEMENT PROTOCOL--RIGID BODY
3RIGID BODY FITREALCross correlationREFINEMENT PROTOCOL--RIGID BODY
4RIGID BODY FITREALCross correlationREFINEMENT PROTOCOL--RIGID BODY
5RIGID BODY FITREALCross correlationREFINEMENT PROTOCOL--RIGID BODY
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-ID
13E1Y

3e1y

A1
23E1Y

3e1y

F1
32KTU

2ktu

A2
41R5B

1r5b

A3
51R5O

1r5o

A4
63VMF

3vmf

A5
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms6636 2088 0 0 8724

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