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Entry
Database: EMDB / ID: EMD-3040
TitleCryo-EM structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ) and the UGA stop codon
Map dataReconstruction of a mammalian 80S ribosome-nascent chain complex containing the UGA stop codon bound to eRF1(AAQ) and ABCE1
Sample
  • Sample: Affinity-purified 80S ribosome-nascent chain complex containing the UGA stop codon bound to eRF1(AAQ) and ABCE1
  • Complex: 80S 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 / translation release factor complex / cytoplasmic translational termination / translation release factor activity / regulation of translational termination / OAS antiviral response / ribosomal subunit / protein methylation ...negative regulation of endoribonuclease activity / CTPase activity / translation termination factor activity / translation release factor complex / cytoplasmic translational termination / translation release factor activity / regulation of translational termination / OAS antiviral response / ribosomal subunit / protein methylation / translation release factor activity, codon specific / ribosome disassembly / sequence-specific mRNA binding / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / aminoacyl-tRNA hydrolase activity / regulation of G1 to G0 transition / positive regulation of intrinsic apoptotic signaling pathway in response to DNA damage by p53 class mediator / regulation of translation involved in cellular response to UV / protein-DNA complex disassembly / positive regulation of DNA damage response, signal transduction by p53 class mediator resulting in transcription of p21 class mediator / mammalian oogenesis stage / G1 to G0 transition / activation-induced cell death of T cells / Protein hydroxylation / positive regulation of signal transduction by p53 class mediator / ubiquitin ligase inhibitor activity / phagocytic cup / Eukaryotic Translation Termination / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / ribosomal small subunit binding / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / TOR signaling / endoribonuclease inhibitor activity / T cell proliferation involved in immune response / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / ribosomal small subunit export from nucleus / erythrocyte development / translation regulator activity / cellular response to actinomycin D / ribosomal subunit export from nucleus / cytosolic ribosome / translational termination / rough endoplasmic reticulum / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / gastrulation / MDM2/MDM4 family protein binding / translational initiation / DNA damage response, signal transduction by p53 class mediator resulting in cell cycle arrest / class I DNA-(apurinic or apyrimidinic site) endonuclease activity / DNA-(apurinic or apyrimidinic site) lyase / negative regulation of ubiquitin-dependent protein catabolic process / rescue of stalled ribosome / ribonucleoside triphosphate phosphatase activity / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / 90S preribosome / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA / ribosomal large subunit biogenesis / cellular response to leukemia inhibitory factor / maturation of SSU-rRNA / positive regulation of translation / small-subunit processome / positive regulation of apoptotic signaling pathway / protein kinase C binding / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / positive regulation of protein-containing complex assembly / placenta development / cellular response to gamma radiation / mRNA 5'-UTR binding / Regulation of expression of SLITs and ROBOs / transcription coactivator binding / cytoplasmic ribonucleoprotein granule / modification-dependent protein catabolic process / spindle / G1/S transition of mitotic cell cycle / rRNA processing / protein tag activity / Interferon alpha/beta signaling / rhythmic process / positive regulation of canonical Wnt signaling pathway / ribosome biogenesis / ribosome binding / glucose homeostasis / regulation of translation / ribosomal small subunit biogenesis / ribosomal small subunit assembly / 4 iron, 4 sulfur cluster binding / small ribosomal subunit / small ribosomal subunit rRNA binding / cell body / T cell differentiation in thymus / 5S rRNA binding / large ribosomal subunit rRNA binding / perikaryon / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / mitochondrial inner membrane / tRNA binding / cytoplasmic translation / postsynaptic density
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 / Ubiquitin-like protein FUBI / Ribosomal protein L6, N-terminal / Ribosomal protein L6, N-terminal domain / Ribosomal protein L2, archaeal-type / Ribosomal protein L28e / Ribosomal L15/L27a, N-terminal / Ribosomal protein L23 / Ribosomal L28e/Mak16 / Ribosomal L28e protein family / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / : / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / Ribosomal protein S12e signature. / Ribosomal protein S12e / TRASH domain / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Small (40S) ribosomal subunit Asc1/RACK1 / Ribosomal protein S21e / Ribosomal protein S21e superfamily / Ribosomal protein S21e / Ribosomal protein L29e / Ribosomal L29e protein family / 40S Ribosomal protein S10 / S27a-like superfamily / Ribosomal protein S10, eukaryotic/archaeal / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal protein S25 / S25 ribosomal protein / : / Ribosomal protein S17e, conserved site / Ribosomal protein S17e signature. / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S27a / Ribosomal protein S8e subdomain, eukaryotes / Ribosomal protein S30 / Ribosomal protein S30 / 40S ribosomal protein S29/30S ribosomal protein S14 type Z / Ribosomal protein L27e, conserved site / Ribosomal protein L27e signature. / Ribosomal protein S7e signature. / Ribosomal protein L44e signature. / Ribosomal protein L10e, conserved site / Ribosomal protein L10e signature. / Ribosomal protein S3, eukaryotic/archaeal / Ribosomal protein L10e / Ribosomal protein S19e / Ribosomal protein S3Ae, conserved site / Ribosomal protein S19e / Ribosomal protein S3Ae signature. / Ribosomal_S19e / Ribosomal protein S27e signature. / Ribosomal protein S4e, N-terminal, conserved site / Ribosomal protein S4e signature. / 40S ribosomal protein S4, C-terminal domain / 40S ribosomal protein S4 C-terminus / : / Ribosomal protein S19A/S15e / Ribosomal protein S8e, conserved site / Ribosomal protein S8e signature. / Ribosomal protein L24e, conserved site / Ribosomal protein L24e signature. / Ribosomal protein L44e / Ribosomal protein L44 / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein S17e / Ribosomal protein S17e-like superfamily / Ribosomal S17 / Ribosomal protein L6e signature. / Ribosomal protein S6, eukaryotic / 40S ribosomal protein S1/3, eukaryotes / Ribosomal protein S4e, N-terminal / RS4NT (NUC023) domain / Ribosomal S24e conserved site / Ribosomal protein S24e signature.
Similarity search - Domain/homology
Small ribosomal subunit protein eS32 / 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 ...Small ribosomal subunit protein eS32 / 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 / Small ribosomal subunit protein uS11 / 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 / Small ribosomal subunit protein eS10 / 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 / Large ribosomal subunit protein eL32 / 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: 3.83 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-3jai
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_3040.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 UGA 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-0.48768422 - 0.71897972
Average (Standard dev.)0.00119808 (±0.02276708)
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-0.4880.7190.001

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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 UGA stop codon bound to eRF1(AAQ) and ABCE1
Components
  • Sample: Affinity-purified 80S ribosome-nascent chain complex containing the UGA stop codon bound to eRF1(AAQ) and ABCE1
  • Complex: 80S 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 UGA 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:
UCAAAGUUUG AG

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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
Specialist opticsEnergy filter - Name: FEI
DetailsAutomated data acquisition using EPU (FEI)
DateApr 17, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 1472 / Average electron dose: 30 e/Å2
Details: Every image is the average of 17 frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.6 µm / Nominal defocus min: 1.7 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
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: 3.83 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION / Number images used: 22058
FSC plot (resolution estimation)

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Atomic model buiding 1

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

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

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Atomic model buiding 2

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

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

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Atomic model buiding 3

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

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

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Atomic model buiding 4

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

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

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Atomic model buiding 5

Initial modelPDB ID:
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-3jai:
Structure of a mammalian ribosomal termination complex with ABCE1, eRF1(AAQ), and the UGA stop codon

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