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- EMDB-3039: Cryo-EM structure of a mammalian ribosomal termination complex wi... -

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Entry
Database: EMDB / ID: EMD-3039
TitleCryo-EM structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ) and the UAG stop codon
Map dataReconstruction of a mammalian 80S ribosome-nascent chain complex containing the UAG stop codon bound to eRF1(AAQ) and ABCE1
Sample
  • Sample: Affinity-purified 80S ribosome-nascent chain complex containing the UAG stop codon bound to eRF1(AAQ) and ABCE1
  • Complex: 80S ribosomeEukaryotic ribosome
  • Protein or peptide: eukaryotic release factor 1, G183A, G184A
  • Protein or peptide: ATP binding cassette E1
  • Protein or peptide: Sec61-beta
  • RNA: messenger RNA
  • RNA: transfer RNA
Keywordsribosome / translation termination / release factor / stop codon / decoding
Function / homology
Function and homology information


negative regulation of endoribonuclease activity / CTPase activity / translation termination factor activity / cytoplasmic translational termination / translation release factor complex / OAS antiviral response / regulation of translational termination / translation release factor activity / ribosome disassembly / translation release factor activity, codon specific ...negative regulation of endoribonuclease activity / CTPase activity / translation termination factor activity / cytoplasmic translational termination / translation release factor complex / OAS antiviral response / regulation of translational termination / translation release factor activity / ribosome disassembly / translation release factor activity, codon specific / protein methylation / ribosomal subunit / sequence-specific mRNA binding / aminoacyl-tRNA hydrolase activity / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / ribosomal subunit export from nucleus / Protein hydroxylation / ribosomal small subunit binding / Eukaryotic Translation Termination / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / endoribonuclease inhibitor activity / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / rough endoplasmic reticulum / rescue of stalled ribosome / translational termination / translational initiation / DNA-(apurinic or apyrimidinic site) lyase / ribonucleoside triphosphate phosphatase activity / cytosolic ribosome / : / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / Regulation of expression of SLITs and ROBOs / cytosolic small ribosomal subunit / Interferon alpha/beta signaling / ribosome binding / ribosome biogenesis / regulation of translation / 4 iron, 4 sulfur cluster binding / small ribosomal subunit / 5S rRNA binding / cytoplasmic translation / rRNA binding / ribosome / mitochondrial matrix / structural constituent of ribosome / translation / iron ion binding / ribonucleoprotein complex / GTPase activity / nucleolus / ATP hydrolysis activity / mitochondrion / RNA binding / ATP binding / membrane / cytosol / cytoplasm
Similarity search - Function
RLI, domain 1 / RLI1 / RNase L inhibitor RLI-like, possible metal-binding domain / Possible Fer4-like domain in RNase L inhibitor, RLI / Peptide chain release factor eRF1/aRF1 / eRF1, domain 1 / eRF1 domain 2 / eRF1 domain 2 / eRF1 domain 1 / eRF1 domain 1/Pelota-like ...RLI, domain 1 / RLI1 / RNase L inhibitor RLI-like, possible metal-binding domain / Possible Fer4-like domain in RNase L inhibitor, RLI / 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 / 4Fe-4S binding domain / 60S acidic ribosomal protein P0 / Ribosomal protein L6, N-terminal / Ribosomal protein L6, N-terminal domain / : / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / TRASH domain / Ribosomal protein L29e / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / Ribosomal L29e protein family / Ribosomal protein S12e signature. / Ribosomal protein L24e, conserved site / Ribosomal protein S19e signature. / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / 40S Ribosomal protein S10 / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal Protein L6, KOW domain / 60S ribosomal protein L6E / Ribosomal protein S17e signature. / Ribosomal protein L44e signature. / Ribosomal protein L27e signature. / Ribosomal protein L10e signature. / Ribosomal protein S7e signature. / Ribosomal protein L6e / Ribosomal protein S3Ae signature. / Ribosomal protein S27e signature. / Ribosomal protein S4e signature. / Ribosomal protein S8e signature. / Ribosomal protein L24e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein L34e signature. / Ribosomal protein L6e signature. / Ribosomal S24e conserved site / Ribosomal protein S24e signature. / Ribosomal protein L36e signature. / Ribosomal protein L39e signature. / Ribosomal protein L6, conserved site-2 / Ribosomal protein L24e-related / Ribosomal protein L24e/L24 superfamily / Ribosomal protein S24e / Ribosomal protein S24e / Ribosomal protein S6e signature. / Ribosomal protein L35Ae signature. / Ribosomal protein S28e signature. / Ribosomal protein L24e / Ribosomal protein L31e signature. / Ribosomal protein L14e domain / Ribosomal protein L14 / Ribosomal protein L5 eukaryotic/L18 archaeal / Ribosomal large subunit proteins 60S L5, and 50S L18 / Ribosomal protein L32e, conserved site / Ribosomal protein L32e signature. / Ribosomal protein L14 / Ribosomal protein L6 signature 2. / Ribosomal protein L14, KOW motif / Ribosomal protein L1e signature. / Ribosomal protein L15e signature. / Ribosomal protein L21e signature. / Ribosomal protein L37e signature. / Ribosomal protein L32e / Ribosomal protein L32e superfamily / Ribosomal protein L32 / Ribosomal_L32e / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain / Ribosomal protein L10P / Ribosomal protein L10 / Ribosomal protein L11 signature. / 50S ribosomal protein L30e-like / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / Ubiquitin domain signature. / Ribosomal protein L23 signature.
Similarity search - Domain/homology
60S ribosomal protein L41 / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein uS4 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL33 / Small ribosomal subunit protein eS12 / ATP binding cassette subfamily E member 1 / Large ribosomal subunit protein eL29 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein eL31 ...60S ribosomal protein L41 / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein uS4 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL33 / Small ribosomal subunit protein eS12 / ATP binding cassette subfamily E member 1 / Large ribosomal subunit protein eL29 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein eL31 / Large ribosomal subunit protein eL21 / Large ribosomal subunit protein uL29 / Small ribosomal subunit protein uS10 / Small ribosomal subunit protein RACK1 / Ubiquitin-ribosomal protein eS31 fusion protein / Large ribosomal subunit protein uL11 / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein uL24 / Small ribosomal subunit protein eS1 / Large ribosomal subunit protein eL8 / Small ribosomal subunit protein eS7 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein eL43 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein eL39 / Small ribosomal subunit protein uS12 / Small ribosomal subunit protein uS13 / Large ribosomal subunit protein eL42 / Large ribosomal subunit protein eL15 / Ribosomal protein S14 / 40S ribosomal protein S24 / Large ribosomal subunit protein uL14 / Ubiquitin-like FUBI-ribosomal protein eS30 fusion protein / Small ribosomal subunit protein eS25 / Small ribosomal subunit protein uS7 / 60S ribosomal protein L6 / Small ribosomal subunit protein uS8 / Small ribosomal subunit protein eS28 / Small ribosomal subunit protein eS8 / Small ribosomal subunit protein eS4 / Large ribosomal subunit protein uL3 / TRASH domain-containing protein / Small ribosomal subunit protein eS6 / Small ribosomal subunit protein eS21 / Small ribosomal subunit protein eS19 / Large ribosomal subunit protein eL29 / Small ribosomal subunit protein uS3 / Plectin/eS10 N-terminal domain-containing protein / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein eL36 / Small ribosomal subunit protein eS17 / Large ribosomal subunit protein uL5 / Ribosomal protein L32 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL6 / Large ribosomal subunit protein eL27 / Small ribosomal subunit protein eS27 / Small ribosomal subunit protein uS19 / 60S acidic ribosomal protein P0 / Large ribosomal subunit protein eL28 / Small ribosomal subunit protein uS14 / Large ribosomal subunit protein eL34 / ATP-binding cassette sub-family E member 1 / Eukaryotic peptide chain release factor subunit 1 / Large ribosomal subunit protein eL14 / Large ribosomal subunit protein eL37
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Homo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.45 Å
AuthorsBrown A / Shao S / Murray J / Hegde RS / Ramakrishnan V
CitationJournal: Nature / Year: 2015
Title: Structural basis for stop codon recognition in eukaryotes.
Authors: Alan Brown / Sichen Shao / Jason Murray / Ramanujan S Hegde / V Ramakrishnan /
Abstract: Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal ...Termination of protein synthesis occurs when a translating ribosome encounters one of three universally conserved stop codons: UAA, UAG or UGA. Release factors recognize stop codons in the ribosomal A-site to mediate release of the nascent chain and recycling of the ribosome. Bacteria decode stop codons using two separate release factors with differing specificities for the second and third bases. By contrast, eukaryotes rely on an evolutionarily unrelated omnipotent release factor (eRF1) to recognize all three stop codons. The molecular basis of eRF1 discrimination for stop codons over sense codons is not known. Here we present cryo-electron microscopy (cryo-EM) structures at 3.5-3.8 Å resolution of mammalian ribosomal complexes containing eRF1 interacting with each of the three stop codons in the A-site. Binding of eRF1 flips nucleotide A1825 of 18S ribosomal RNA so that it stacks on the second and third stop codon bases. This configuration pulls the fourth position base into the A-site, where it is stabilized by stacking against G626 of 18S rRNA. Thus, eRF1 exploits two rRNA nucleotides also used during transfer RNA selection to drive messenger RNA compaction. In this compacted mRNA conformation, stop codons are favoured by a hydrogen-bonding network formed between rRNA and essential eRF1 residues that constrains the identity of the bases. These results provide a molecular framework for eukaryotic stop codon recognition and have implications for future studies on the mechanisms of canonical and premature translation termination.
History
DepositionJun 7, 2015-
Header (metadata) releaseJul 15, 2015-
Map releaseAug 12, 2015-
UpdateDec 23, 2015-
Current statusDec 23, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3jah
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_3039.tif

Downloads & links

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Map

FileDownload / File: emd_3039.map.gz / Format: CCP4 / Size: 276 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of a mammalian 80S ribosome-nascent chain complex containing the UAG stop codon bound to eRF1(AAQ) and ABCE1
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.315375 - 0.52816588
Average (Standard dev.)0.00124731 (±0.01692608)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions420420420
Spacing420420420
CellA=B=C: 562.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z420420420
origin x/y/z0.0000.0000.000
length x/y/z562.800562.800562.800
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS420420420
D min/max/mean-0.3150.5280.001

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Supplemental data

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Sample components

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Entire : Affinity-purified 80S ribosome-nascent chain complex containing t...

EntireName: Affinity-purified 80S ribosome-nascent chain complex containing the UAG stop codon bound to eRF1(AAQ) and ABCE1
Components
  • Sample: Affinity-purified 80S ribosome-nascent chain complex containing the UAG stop codon bound to eRF1(AAQ) and ABCE1
  • Complex: 80S ribosomeEukaryotic ribosome
  • Protein or peptide: eukaryotic release factor 1, G183A, G184A
  • Protein or peptide: ATP binding cassette E1
  • Protein or peptide: Sec61-beta
  • RNA: messenger RNA
  • RNA: transfer RNA

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Supramolecule #1000: Affinity-purified 80S ribosome-nascent chain complex containing t...

SupramoleculeName: Affinity-purified 80S ribosome-nascent chain complex containing the UAG stop codon bound to eRF1(AAQ) and ABCE1
type: sample / ID: 1000 / Number unique components: 6
Molecular weightTheoretical: 2.1 MDa

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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: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Cell: reticulocyte / Location in cell: cytosol
Molecular weightTheoretical: 2 MDa

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Macromolecule #1: eukaryotic release factor 1, G183A, G184A

MacromoleculeName: eukaryotic release factor 1, G183A, G184A / type: protein_or_peptide / ID: 1 / Name.synonym: eRF1(AAQ) / Number of copies: 1 / Recombinant expression: Yes
Source (natural)Organism: Homo sapiens (human) / synonym: Human
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant plasmid: pRSETA
SequenceUniProtKB: Eukaryotic peptide chain release factor subunit 1

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Macromolecule #2: ATP binding cassette E1

MacromoleculeName: ATP binding cassette E1 / type: protein_or_peptide / ID: 2 / Name.synonym: ABCE1, Rli1 / Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Location in cell: cytosol
SequenceUniProtKB: ATP-binding cassette sub-family E member 1

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Macromolecule #4: Sec61-beta

MacromoleculeName: Sec61-beta / type: protein_or_peptide / ID: 4 / Details: in vitro translated peptide sequence / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human

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Macromolecule #3: messenger RNA

MacromoleculeName: messenger RNA / type: rna / ID: 3 / Name.synonym: mRNA
Details: in vitro transcribed mRNA sequence containing UAA stop codon
Classification: OTHER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Homo sapiens (human) / synonym: Human
SequenceString:
UCAAAGUUUA GG

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Macromolecule #5: transfer RNA

MacromoleculeName: transfer RNA / type: rna / ID: 5 / Name.synonym: tRNA / Classification: OTHER / Structure: OTHER / Synthetic?: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit
Molecular weightTheoretical: 20 KDa

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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: 7.4 / Details: 50 mM Hepes, 100 mM KAc, 5 mM MgAc2, 1 mM DTT
GridDetails: R2/2 400 mesh Cu grids with thin continuous carbon support, glow discharged
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III / Method: 30 sec wait time, blot for 3 sec before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 59000
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DetailsAutomated data acquisition using EPU (FEI)
DateFeb 25, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1601 / Average electron dose: 30 e/Å2
Details: Every image is the average of 17 frames recorded by the direct electron detector
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.45 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION / Number images used: 20515
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3j92

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

PDB-3jah:
Structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ), and the UAG stop codon

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Atomic model buiding 2

Initial modelPDB ID:

3j7p

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

PDB-3jah:
Structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ), and the UAG stop codon

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Atomic model buiding 3

Initial modelPDB ID:

1dt9

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

PDB-3jah:
Structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ), and the UAG stop codon

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Atomic model buiding 4

Initial modelPDB ID:

3bk7

SoftwareName: Chimera, Coot
DetailsSequence was modified in Coot to agree with rabbit sequence
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT
Output model

PDB-3jah:
Structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ), and the UAG stop codon

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Atomic model buiding 5

Initial modelPDB ID:

4v51

SoftwareName: Chimera, Coot
DetailsSequence was modified in Coot to agree with the most prevalent tRNA sequence for each particular codon
RefinementSpace: RECIPROCAL / Protocol: FLEXIBLE FIT
Output model

PDB-3jah:
Structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ), and the UAG stop codon

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