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- EMDB-2421: Molecular architecture of the 80S-eIF5B-Met-itRNAMet Eukaryotic T... -

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Basic information

Entry
Database: EMDB / ID: EMD-2421
TitleMolecular architecture of the 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex
Map dataReconstruction of 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex with tRNA in the P/E-site and only density for the G domain and domain II of eIF5B
Sample
  • Sample: 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex with tRNA in the P/E-site and only density for the G domain and domain II
  • Complex: 80S-Met-itRNAMet Eukaryotic Translation Initiation Complex
  • Protein or peptide: eIF5B
KeywordsRibosome initiation complex / initiator factor eiF5B / cryo EM / single particle analysis
Function / homology
Function and homology information


triplex DNA binding / ribosome hibernation / translation elongation factor binding / regulation of translational initiation in response to stress / Platelet degranulation / protein-synthesizing GTPase / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, LSU-rRNA,5S) / formation of cytoplasmic translation initiation complex / eukaryotic 48S preinitiation complex / negative regulation of glucose mediated signaling pathway ...triplex DNA binding / ribosome hibernation / translation elongation factor binding / regulation of translational initiation in response to stress / Platelet degranulation / protein-synthesizing GTPase / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, LSU-rRNA,5S) / formation of cytoplasmic translation initiation complex / eukaryotic 48S preinitiation complex / negative regulation of glucose mediated signaling pathway / negative regulation of translational frameshifting / Negative regulators of DDX58/IFIH1 signaling / positive regulation of translational fidelity / Protein methylation / RMTs methylate histone arginines / mTORC1-mediated signalling / Protein hydroxylation / ribosome-associated ubiquitin-dependent protein catabolic process / GDP-dissociation inhibitor activity / regulation of translational initiation / positive regulation of nuclear-transcribed mRNA catabolic process, deadenylation-dependent decay / pre-mRNA 5'-splice site binding / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition / translational elongation / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / preribosome, small subunit precursor / response to cycloheximide / telomeric DNA binding / mRNA destabilization / Major pathway of rRNA processing in the nucleolus and cytosol / 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 / L13a-mediated translational silencing of Ceruloplasmin expression / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / endonucleolytic cleavage to generate mature 3'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / G-protein alpha-subunit binding / TOR signaling / positive regulation of protein kinase activity / protein-RNA complex assembly / regulation of translational fidelity / Ub-specific processing proteases / ribosomal small subunit export from nucleus / translation regulator activity / ribosomal subunit export from nucleus / translational termination / 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) / translation repressor activity / DNA-(apurinic or apyrimidinic site) endonuclease activity / translation initiation factor binding / translational initiation / translation initiation factor activity / cellular response to amino acid starvation / ribosome assembly / telomere maintenance / rescue of stalled ribosome / 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) / cytosolic ribosome assembly / maturation of LSU-rRNA / ribosomal large subunit biogenesis / maturation of SSU-rRNA / small-subunit processome / positive regulation of apoptotic signaling pathway / protein kinase C binding / maintenance of translational fidelity / ribosomal large subunit assembly / cytoplasmic stress granule / modification-dependent protein catabolic process / rRNA processing / protein tag activity / ribosome biogenesis / ribosome binding / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic small ribosomal subunit / cytosolic large ribosomal subunit / cytoplasmic translation / rRNA binding / negative regulation of translation / ribosome / protein ubiquitination / structural constituent of ribosome / translation / positive regulation of protein phosphorylation / G protein-coupled receptor signaling pathway / negative regulation of gene expression / response to antibiotic / GTPase activity
Similarity search - Function
Elongation factor Tu-type domain / Elongation factor Tu domain 4 / Stm1-like, N-terminal / Stm1 / Hyaluronan/mRNA-binding protein / Hyaluronan / mRNA binding family / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily ...Elongation factor Tu-type domain / Elongation factor Tu domain 4 / Stm1-like, N-terminal / Stm1 / Hyaluronan/mRNA-binding protein / Hyaluronan / mRNA binding family / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / 60s Acidic ribosomal protein / 60S acidic ribosomal protein P0 / 50S ribosomal protein L10, insertion domain superfamily / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / : / Ribosomal protein S26e signature. / : / Ribosomal protein L41 / Ribosomal protein L41 / Ribosomal protein S26e / Ribosomal protein S26e superfamily / Ribosomal protein S26e / Ribosomal protein S21e, conserved site / Ribosomal protein S21e signature. / Ribosomal protein S12e signature. / Ribosomal protein S12e / Ribosomal protein S5, eukaryotic/archaeal / Ribosomal protein S19e, conserved site / Ribosomal protein S19e signature. / Small (40S) ribosomal subunit Asc1/RACK1 / Ribosomal protein S2, eukaryotic / 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 / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Plectin/S10, N-terminal / Plectin/S10 domain / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein S25 / S25 ribosomal protein / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal L38e protein family / Ribosomal protein S2, eukaryotic/archaeal / : / 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 L13e / Ribosomal protein L13e / Ribosomal protein L19, eukaryotic / Ribosomal protein S19e / Ribosomal protein S3Ae, conserved site / Ribosomal protein S19e / Ribosomal protein S3Ae signature. / Ribosomal_S19e / Ribosomal protein S27e signature. / 60S ribosomal protein L18a/ L20, eukaryotes / 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 L19/L19e conserved site / Ribosomal protein L44 / Ribosomal protein L19e signature. / Ribosomal protein L34e, conserved site / Ribosomal protein L34e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein S17e
Similarity search - Domain/homology
Small ribosomal subunit protein uS4A / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein eS17A / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A ...Small ribosomal subunit protein uS4A / Large ribosomal subunit protein uL15 / Small ribosomal subunit protein eS17A / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / 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 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS15 / Ubiquitin-ribosomal protein eS31 fusion protein / Small ribosomal subunit protein uS11A / Small ribosomal subunit protein eS19A / Small ribosomal subunit protein eS21A / Small ribosomal subunit protein uS8A / 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 eL20B / Large ribosomal subunit protein eL42A / Small ribosomal subunit protein uS12A / Small ribosomal subunit protein eS24A / Small ribosomal subunit protein eS30A / Small ribosomal subunit protein eS4A / Small ribosomal subunit protein eS6A / Small ribosomal subunit protein eS8A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL2B / Small ribosomal subunit protein uS17A / Large ribosomal subunit protein eL18A / Small ribosomal subunit protein uS9A / Small ribosomal subunit protein uS13A / Large ribosomal subunit protein eL19 / Large ribosomal subunit protein uL29B / Small ribosomal subunit protein eS32B / Large ribosomal subunit protein uL4A / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein uL18 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein uL13A / Small ribosomal subunit protein eS7A / Small ribosomal subunit protein uS2A / Small ribosomal subunit protein eS1A / Small ribosomal subunit protein eS27A / Small ribosomal subunit protein RACK1 / Large ribosomal subunit protein eL32 / Small ribosomal subunit protein uS10 / Large ribosomal subunit protein eL14B / Suppressor protein STM1 / Eukaryotic translation initiation factor 5B / Small ribosomal subunit protein eS26A / Small ribosomal subunit protein uS14A / Large ribosomal subunit protein uL16 / Small ribosomal subunit protein eS12 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Large ribosomal subunit protein eL34A / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Small ribosomal subunit protein eS10A / Large ribosomal subunit protein eL13A / Small ribosomal subunit protein eS25A / Small ribosomal subunit protein eS28A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 4.3 Å
AuthorsFernandez IS / Bai XC / Hussain T / Kelley AC / Lorsch JR / Ramakrishnan V / Scheres SHW
CitationJournal: Science / Year: 2013
Title: Molecular architecture of a eukaryotic translational initiation complex.
Authors: Israel S Fernández / Xiao-Chen Bai / Tanweer Hussain / Ann C Kelley / Jon R Lorsch / V Ramakrishnan / Sjors H W Scheres /
Abstract: The last step in eukaryotic translational initiation involves the joining of the large and small subunits of the ribosome, with initiator transfer RNA (Met-tRNA(i)(Met)) positioned over the start ...The last step in eukaryotic translational initiation involves the joining of the large and small subunits of the ribosome, with initiator transfer RNA (Met-tRNA(i)(Met)) positioned over the start codon of messenger RNA in the P site. This step is catalyzed by initiation factor eIF5B. We used recent advances in cryo-electron microscopy (cryo-EM) to determine a structure of the eIF5B initiation complex to 6.6 angstrom resolution from <3% of the population, comprising just 5143 particles. The structure reveals conformational changes in eIF5B, initiator tRNA, and the ribosome that provide insights into the role of eIF5B in translational initiation. The relatively high resolution obtained from such a small fraction of a heterogeneous sample suggests a general approach for characterizing the structure of other dynamic or transient biological complexes.
History
DepositionJul 19, 2013-
Header (metadata) releaseAug 7, 2013-
Map releaseNov 20, 2013-
UpdateNov 27, 2013-
Current statusNov 27, 2013Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4v8y
  • Surface level: 0.25
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2421.jpg

Downloads & links

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Map

FileDownload / File: emd_2421.map.gz / Format: CCP4 / Size: 51.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex with tRNA in the P/E-site and only density for the G domain and domain II of eIF5B
Voxel sizeX=Y=Z: 1.77 Å
Density
Contour LevelBy AUTHOR: 0.25 / Movie #1: 0.25
Minimum - Maximum-0.93000621 - 1.37437451
Average (Standard dev.)0.00303775 (±0.08476789)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-120-120-120
Dimensions240240240
Spacing240240240
CellA=B=C: 424.8 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.771.771.77
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z424.800424.800424.800
α/β/γ90.00090.00090.000
start NX/NY/NZ00-40
NX/NY/NZ555581
MAP C/R/S123
start NC/NR/NS-120-120-120
NC/NR/NS240240240
D min/max/mean-0.9301.3740.003

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Supplemental data

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Sample components

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Entire : 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex ...

EntireName: 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex with tRNA in the P/E-site and only density for the G domain and domain II
Components
  • Sample: 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex with tRNA in the P/E-site and only density for the G domain and domain II
  • Complex: 80S-Met-itRNAMet Eukaryotic Translation Initiation Complex
  • Protein or peptide: eIF5B

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Supramolecule #1000: 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex ...

SupramoleculeName: 80S-eIF5B-Met-itRNAMet Eukaryotic Translation Initiation Complex with tRNA in the P/E-site and only density for the G domain and domain II
type: sample / ID: 1000 / Number unique components: 2
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 MDa

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Supramolecule #1: 80S-Met-itRNAMet Eukaryotic Translation Initiation Complex

SupramoleculeName: 80S-Met-itRNAMet Eukaryotic Translation Initiation Complex
type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 MDa

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Macromolecule #1: eIF5B

MacromoleculeName: eIF5B / type: protein_or_peptide / ID: 1 / Details: Point mutation T439A / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.3 mg/mL
BufferpH: 7.2
Details: 3mM Hepes-KOH, 6.6 mM Tris-acetate pH 7.2, 3 mM NH4Cl, 6.6 mM NH4-acetate, 48 mM K-acetate, 4 mM Mg-acetate, 2.4 mM DTT
StainingType: NEGATIVE / Details: cryo-EM
GridDetails: Quantifoil grids (2/2) with 3 nm thin carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 90 K / Instrument: FEI VITROBOT MARK II / Method: Blot 2.5 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureMin: 80 K / Max: 90 K / Average: 85 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 59,000 times magnification
DateJan 15, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON I (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 1012 / Average electron dose: 16 e/Å2
Details: Every image is the average of 16 frames recorded by the direct electron detector
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 79096 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2 mm / Nominal defocus max: 3.9 µm / Nominal defocus min: 1.9 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsUse a newly developed statistical movie processing approach to compensate for beam-induced movement. Use a newly developed statistical movie processing to compensate for beam-induced movement.
CTF correctionDetails: Each particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION
Details: Use a newly developed statistical movie processing approach to compensate for beam-induced movement.
Number images used: 40729

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