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- EMDB-3227: Cryo-EM map of a native 80S-ribosome-eIF-5A complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3227
TitleCryo-EM map of a native 80S-ribosome-eIF-5A complex
Map dataRibosome reconstruction
Sample
  • Sample: Native 80S ribosome-eIF-5A complex from yeast
  • Complex: Ribosome
  • Protein or peptide: eIF-5AEIF5A
KeywordsTranslation
Function / homology
Function and homology information


positive regulation of cytoplasmic translational elongation through polyproline stretches / Hypusine synthesis from eIF5A-lysine / CAT tailing / translational frameshifting / positive regulation of translational termination / positive regulation of translational elongation / pre-mRNA 5'-splice site binding / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translational elongation / response to cycloheximide ...positive regulation of cytoplasmic translational elongation through polyproline stretches / Hypusine synthesis from eIF5A-lysine / CAT tailing / translational frameshifting / positive regulation of translational termination / positive regulation of translational elongation / pre-mRNA 5'-splice site binding / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translational elongation / response to cycloheximide / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Formation of a pool of free 40S subunits / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / negative regulation of mRNA splicing, via spliceosome / protein-RNA complex assembly / preribosome, large subunit precursor / positive regulation of translational initiation / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal large subunit export from nucleus / regulation of translational fidelity / rescue of stalled ribosome / translational termination / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / translation elongation factor activity / maturation of LSU-rRNA / ribosomal large subunit biogenesis / translation initiation factor activity / maintenance of translational fidelity / macroautophagy / modification-dependent protein catabolic process / protein tag activity / rRNA processing / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / ribosome binding / ribosome biogenesis / 5S rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / negative regulation of translation / rRNA binding / protein ubiquitination / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / ubiquitin protein ligase binding / perinuclear region of cytoplasm / mitochondrion / RNA binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
: / Translation initiation factor 5A-like, N-terminal / Translation elongation factor, IF5A, hypusine site / Eukaryotic initiation factor 5A hypusine signature. / Eukaryotic elongation factor 5A hypusine, DNA-binding OB fold / Translation elongation factor IF5A-like / Translation elongation factor, IF5A C-terminal / Eukaryotic elongation factor 5A hypusine, DNA-binding OB fold / : / Ribosomal protein L29e ...: / Translation initiation factor 5A-like, N-terminal / Translation elongation factor, IF5A, hypusine site / Eukaryotic initiation factor 5A hypusine signature. / Eukaryotic elongation factor 5A hypusine, DNA-binding OB fold / Translation elongation factor IF5A-like / Translation elongation factor, IF5A C-terminal / Eukaryotic elongation factor 5A hypusine, DNA-binding OB fold / : / Ribosomal protein L29e / Ribosomal protein L10e, conserved site / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein L10e / Ribosomal protein L27e, conserved site / Ribosomal L29e protein family / Ribosomal protein L24e, conserved site / Ribosomal protein L34e, conserved site / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein L44e / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal protein L27e / Ribosomal protein L27e superfamily / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L38e protein family / Ribosomal L22e protein family / Ribosomal protein L23/L25, N-terminal / 60S ribosomal protein L35 / Ribosomal protein L35Ae, conserved site / Ribosomal protein L30e, conserved site / Ribosomal protein L44 / Ribosomal protein L34Ae / Ribosomal protein L23, N-terminal domain / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal L27e protein family / Ribosomal Protein L6, KOW domain / Ribosomal protein L30/YlxQ / Ribosomal protein L13e / Ribosomal protein L13e / Ribosomal protein L34e / Ribosomal protein L31e, conserved site / Ribosomal protein L37ae / 60S ribosomal protein L6E / Ribosomal protein L19, eukaryotic / Ribosomal protein L35A / Ribosomal protein L36e / Ribosomal protein L36e domain superfamily / Ribosomal L40e family / Ribosomal protein L36e / Ribosomal protein L35A superfamily / Ribosomal protein L44e signature. / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / Ribosomal protein L7A/L8 / Ribosomal protein L27e signature. / Ribosomal protein L10e signature. / 60S ribosomal protein L4, C-terminal domain / 60S ribosomal protein L18a/ L20, eukaryotes / Ribosomal protein L7, eukaryotic / Ribosomal protein L6e / Ribosomal protein L30, N-terminal / Ribosomal protein L6, conserved site-2 / Ribosomal L30 N-terminal domain / Ribosomal protein L19/L19e conserved site / Ribosomal L37ae protein family / Ribosomal protein L35Ae / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / Ribosomal protein L39e, conserved site / Ribosomal protein 50S-L18Ae/60S-L20/60S-L18A / 60S ribosomal protein L4 C-terminal domain / Ribosomal protein L19e signature. / Ribosomal proteins 50S-L18Ae/60S-L20/60S-L18A / Ribosomal protein L24e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein L34e signature. / Ribosomal protein L31e / Ribosomal protein L31e domain superfamily / Ribosomal protein L6e signature. / Ribosomal_L31e / Ribosomal protein L15e, conserved site / Ribosomal protein L30e signature 1. / Ribosomal protein L21e / Ribosomal protein L21e, conserved site / Ribosomal protein L21 superfamily / Ribosomal protein L14e domain / Ribosomal protein 60S L18 and 50S L18e / Ribosomal protein L30e signature 2. / 60S ribosomal protein L19 / Ribosomal protein L32e, conserved site / Ribosomal protein L4/L1e, eukaryotic/archaeal, conserved site / Ribosomal protein L37e, conserved site / Ribosomal protein L3, domain 3, archaeal type superfamily / Ribosomal protein L3, archaeal/eukaryotic type / Ribosomal protein L36e signature. / Ribosomal protein L37e
Similarity search - Domain/homology
Large ribosomal subunit protein uL15 / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A ...Large ribosomal subunit protein uL15 / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL29 / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL22A / Large ribosomal subunit protein uL5A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Ubiquitin-ribosomal protein eL40A fusion protein / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein eL42A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL2A / Large ribosomal subunit protein eL18A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Small ribosomal subunit protein eS32A / Large ribosomal subunit protein uL4A / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / Eukaryotic translation initiation factor 5A-1 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL13A / Large ribosomal subunit protein eL14A / Large ribosomal subunit protein eL32 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Large ribosomal subunit protein eL34A / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Large ribosomal subunit protein eL13A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.88 Å
AuthorsSchmidt C / Becker T / Heuer A / Braunger K / Shanmuganathan V / Pech M / m Berninghausen O / Wilson D / Beckmann R
CitationJournal: Nucleic Acids Res / Year: 2016
Title: Structure of the hypusinylated eukaryotic translation factor eIF-5A bound to the ribosome.
Authors: Christian Schmidt / Thomas Becker / André Heuer / Katharina Braunger / Vivekanandan Shanmuganathan / Markus Pech / Otto Berninghausen / Daniel N Wilson / Roland Beckmann /
Abstract: During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the ...During protein synthesis, ribosomes become stalled on polyproline-containing sequences, unless they are rescued in archaea and eukaryotes by the initiation factor 5A (a/eIF-5A) and in bacteria by the homologous protein EF-P. While a structure of EF-P bound to the 70S ribosome exists, structural insight into eIF-5A on the 80S ribosome has been lacking. Here we present a cryo-electron microscopy reconstruction of eIF-5A bound to the yeast 80S ribosome at 3.9 Å resolution. The structure reveals that the unique and functionally essential post-translational hypusine modification reaches toward the peptidyltransferase center of the ribosome, where the hypusine moiety contacts A76 of the CCA-end of the P-site tRNA. These findings would support a model whereby eIF-5A stimulates peptide bond formation on polyproline-stalled ribosomes by stabilizing and orienting the CCA-end of the P-tRNA, rather than by directly contributing to the catalysis.
History
DepositionOct 29, 2015-
Header (metadata) releaseDec 9, 2015-
Map releaseJan 13, 2016-
UpdateMar 16, 2016-
Current statusMar 16, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5gak
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5gak
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD_3227.jpg

Downloads & links

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Map

FileDownload / File: emd_3227.map.gz / Format: CCP4 / Size: 188.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRibosome reconstruction
Voxel sizeX=Y=Z: 1.084 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.24365851 - 0.40369952
Average (Standard dev.)0.0003013 (±0.0218598)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions370370370
Spacing370370370
CellA=B=C: 401.08 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0841.0841.084
M x/y/z370370370
origin x/y/z0.0000.0000.000
length x/y/z401.080401.080401.080
α/β/γ90.00090.00090.000
start NX/NY/NZ-300-64
NX/NY/NZ6161129
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS370370370
D min/max/mean-0.2440.4040.000

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Supplemental data

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Sample components

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Entire : Native 80S ribosome-eIF-5A complex from yeast

EntireName: Native 80S ribosome-eIF-5A complex from yeast
Components
  • Sample: Native 80S ribosome-eIF-5A complex from yeast
  • Complex: Ribosome
  • Protein or peptide: eIF-5AEIF5A

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Supramolecule #1000: Native 80S ribosome-eIF-5A complex from yeast

SupramoleculeName: Native 80S ribosome-eIF-5A complex from yeast / type: sample / ID: 1000 / Details: Sample was obtained from a native pullout / Oligomeric state: One ribosome binds one eIF-5A molecule / Number unique components: 2

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Supramolecule #1: Ribosome

SupramoleculeName: Ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast / Location in cell: Cytoplasm

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Macromolecule #1: eIF-5A

MacromoleculeName: eIF-5A / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 170 KDa
SequenceUniProtKB: Eukaryotic translation initiation factor 5A-1

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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: 20 mM HEPES, pH7.4, 100 mM KOAc, 10 mM MgCl2, 0.01 mg/ml Cycloheximide
GridDetails: Quantifoil R3/3 grids with 2 nM carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / 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: 3.4 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateFeb 25, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Number real images: 3786
Details: The data was provided with the semi-automated software EPU (FEI Company) with a dose of 2.4 electrons per frame for 13 frames in total. The frames were aligned with the Motion Correction software
Bits/pixel: 16
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each Particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.88 Å / Resolution method: OTHER / Software - Name: SIGNATURE, CTFFIND3, SPIDER, RELION / Number images used: 62532
DetailsComputational sorting and classification was done in SPIDER, movie processing and high resolution refinement was done with RELION

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Atomic model buiding 1

Initial modelPDB ID:

4v88

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

PDB-5gak:
Yeast 60S ribosomal subunit with A-site tRNA, P-site tRNA and eIF-5A

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