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- EMDB-3099: Cryo-EM structure of a human translation termination complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3099
TitleCryo-EM structure of a human translation termination complex
Map dataCryo-EM structure of a human translation termination complex
Sample
  • Sample: CMV stalled human 80S ribosome bound to the translation termination factor eRF1
  • Complex: Human 80S ribosomeEukaryotic ribosome
  • Protein or peptide: translation termination factor eRF1
Keywordshuman / 80S / ribosome / eRF1 / translation termination / CMV / stalling
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 / 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 / Major pathway of rRNA processing in the nucleolus and cytosol / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / rough endoplasmic reticulum / translational termination / cytosolic ribosome / Regulation of expression of SLITs and ROBOs / large ribosomal subunit rRNA binding / ribosome binding / cytoplasmic translation / cytosolic large ribosomal subunit / postsynaptic density / structural constituent of ribosome / translation / ribonucleoprotein complex / focal adhesion / nucleolus / RNA binding / extracellular exosome / membrane / nucleus / 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 / 60S ribosomal protein L4, C-terminal domain / 60S ribosomal protein L4 C-terminal domain / Ribosomal protein L4/L1e, eukaryotic/archaeal, conserved site / Ribosomal protein L4, eukaryotic and archaeal type / Ribosomal protein L22/L17, eukaryotic/archaeal / Ribosomal protein L1e signature. / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L11, N-terminal / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11, RNA binding domain / 50S ribosomal protein L30e-like / Ribosomal protein L22/L17, conserved site / Ribosomal protein L22 signature. / Ribosomal protein L22/L17 / Ribosomal protein L22/L17 superfamily / Ribosomal protein L4/L1e / Ribosomal protein L4 domain superfamily / Ribosomal protein L22p/L17e / Ribosomal protein L4/L1 family
Similarity search - Domain/homology
Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein uL4 / Eukaryotic peptide chain release factor subunit 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsMatheisl S / Berninghausen O / Becker T / Beckmann R
CitationJournal: Nucleic Acids Res / Year: 2015
Title: Structure of a human translation termination complex.
Authors: Sarah Matheisl / Otto Berninghausen / Thomas Becker / Roland Beckmann /
Abstract: In contrast to bacteria that have two release factors, RF1 and RF2, eukaryotes only possess one unrelated release factor eRF1, which recognizes all three stop codons of the mRNA and hydrolyses the ...In contrast to bacteria that have two release factors, RF1 and RF2, eukaryotes only possess one unrelated release factor eRF1, which recognizes all three stop codons of the mRNA and hydrolyses the peptidyl-tRNA bond. While the molecular basis for bacterial termination has been elucidated, high-resolution structures of eukaryotic termination complexes have been lacking. Here we present a 3.8 Å structure of a human translation termination complex with eRF1 decoding a UAA(A) stop codon. The complex was formed using the human cytomegalovirus (hCMV) stalling peptide, which perturbs the peptidyltransferase center (PTC) to silence the hydrolysis activity of eRF1. Moreover, unlike sense codons or bacterial stop codons, the UAA stop codon adopts a U-turn-like conformation within a pocket formed by eRF1 and the ribosome. Inducing the U-turn conformation for stop codon recognition rationalizes how decoding by eRF1 includes monitoring geometry in order to discriminate against sense codons.
History
DepositionJul 16, 2015-
Header (metadata) releaseAug 12, 2015-
Map releaseSep 30, 2015-
UpdateOct 28, 2015-
Current statusOct 28, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0009
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0009
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a8l
  • Surface level: 0.0009
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5a8l
  • Surface level: 0.0009
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5a8l
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3099.png

Downloads & links

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Map

FileDownload / File: emd_3099.map.gz / Format: CCP4 / Size: 276 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of a human translation termination complex
Voxel sizeX=Y=Z: 1.062 Å
Density
Contour LevelBy AUTHOR: 0.0008 / Movie #1: 0.0009
Minimum - Maximum-0.00199406 - 0.00342648
Average (Standard dev.)0.00000441 (±0.00026711)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 446.04 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0621.0621.062
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z446.040446.040446.040
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.0020.0030.000

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Supplemental data

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Sample components

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Entire : CMV stalled human 80S ribosome bound to the translation terminati...

EntireName: CMV stalled human 80S ribosome bound to the translation termination factor eRF1
Components
  • Sample: CMV stalled human 80S ribosome bound to the translation termination factor eRF1
  • Complex: Human 80S ribosomeEukaryotic ribosome
  • Protein or peptide: translation termination factor eRF1

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Supramolecule #1000: CMV stalled human 80S ribosome bound to the translation terminati...

SupramoleculeName: CMV stalled human 80S ribosome bound to the translation termination factor eRF1
type: sample / ID: 1000 / Number unique components: 2
Molecular weightExperimental: 4.5 MDa / Theoretical: 4.5 MDa / Method: Sedimentation

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Supramolecule #1: Human 80S ribosome

SupramoleculeName: Human 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Homo sapiens (human) / synonym: Human / Cell: HeLa S3 spinner cells
Molecular weightExperimental: 4.5 MDa / Theoretical: 4.5 MDa

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Macromolecule #1: translation termination factor eRF1

MacromoleculeName: translation termination factor eRF1 / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: HUMAN

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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

BufferpH: 7.5
Details: 20 mM HEPES, 100 mM KOAc, 2.5 mM Mg(OAc)2, 0.25 mM spermidine, 2 mM DTT, 0.06 U/uL RNasin (Ambion), 1/625 EDTA-free complete protease inhibitor (Roche)
GridDetails: 2 nm pre-coated Quantifoil R3/3 holey carbon supported grids
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.7 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateMar 11, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 3
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: defocus groups
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: OTHER / Software - Name: Spider
Details: Since images from microscopy were processed in the absence of spatial frequencies higher than 8 A, a FSC cut-off value of 0.143 was used for average resolution determination of 3.9 A (Scheres and Chen, 2012).
Number images used: 33165

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Atomic model buiding 1

Initial modelPDB ID:

3e1y

SoftwareName: Chimera, Coot
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5a8l:
Human eRF1 and the hCMV nascent peptide in the translation termination complex

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