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- EMDB-2682: Mammalian 80S-HCV-IRES initiation complex with eIF5B PRE-like state -

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Basic information

Entry
Database: EMDB / ID: EMD-2682
TitleMammalian 80S-HCV-IRES initiation complex with eIF5B PRE-like state
Map dataMammalian translation initiation 80S HCV-IRES complex with eIF5B PRE-like state
Sample
  • Sample: Mammalian translation initiation 80S HCV-IRES complex with eIF5B; PRE-like state
  • Complex: 80S ribosome
  • RNA: tRNA
  • RNA: HCV-IRES
  • Protein or peptide: eIF5B
Keywordsribosome / translation initiation / Hepatitis C Virus internal ribosome entry site / eukaryotic initiation factor 5B
Function / homology
Function and homology information


regulation of translational initiation / translation initiation factor activity / ribosome assembly / tRNA binding / GTPase activity / synapse / GTP binding / mitochondrion
Similarity search - Function
Elongation factor Tu-type domain / Elongation factor Tu domain 4 / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain ...Elongation factor Tu-type domain / Elongation factor Tu domain 4 / Translation initiation factor IF- 2, domain 3 / Translation-initiation factor 2 / Translation initiation factor IF- 2 / Translation initiation factor IF-2, domain 3 superfamily / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Eukaryotic translation initiation factor 5B / Eukaryotic translation initiation factor 5B
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit) / Hepatitis C virus
Methodsingle particle reconstruction / cryo EM / Resolution: 8.9 Å
AuthorsYamamoto H / Unbehaun A / Loerke J / Behrmann E / Collier M / Marianne C / Burger J / Mielke T / Spahn CMT
CitationJournal: Nat Struct Mol Biol / Year: 2014
Title: Structure of the mammalian 80S initiation complex with initiation factor 5B on HCV-IRES RNA.
Authors: Hiroshi Yamamoto / Anett Unbehaun / Justus Loerke / Elmar Behrmann / Marianne Collier / Jörg Bürger / Thorsten Mielke / Christian M T Spahn /
Abstract: The universally conserved eukaryotic initiation factor (eIF) 5B, a translational GTPase, is essential for canonical translation initiation. It is also required for initiation facilitated by the ...The universally conserved eukaryotic initiation factor (eIF) 5B, a translational GTPase, is essential for canonical translation initiation. It is also required for initiation facilitated by the internal ribosomal entry site (IRES) of hepatitis C virus (HCV) RNA. eIF5B promotes joining of 60S ribosomal subunits to 40S ribosomal subunits bound by initiator tRNA (Met-tRNAi(Met)). However, the exact molecular mechanism by which eIF5B acts has not been established. Here we present cryo-EM reconstructions of the mammalian 80S-HCV-IRES-Met-tRNAi(Met)-eIF5B-GMPPNP complex. We obtained two substates distinguished by the rotational state of the ribosomal subunits and the configuration of initiator tRNA in the peptidyl (P) site. Accordingly, a combination of conformational changes in the 80S ribosome and in initiator tRNA facilitates binding of the Met-tRNAi(Met) to the 60S P site and redefines the role of eIF5B as a tRNA-reorientation factor.
History
DepositionJun 17, 2014-
Header (metadata) releaseJul 9, 2014-
Map releaseJul 30, 2014-
UpdateJan 27, 2016-
Current statusJan 27, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 2000
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 2000
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-4ujd
  • Surface level: 2000
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2682.png

Downloads & links

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Map

FileDownload / File: emd_2682.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMammalian translation initiation 80S HCV-IRES complex with eIF5B PRE-like state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.26 Å/pix.
x 360 pix.
= 453.6 Å		1.26 Å/pix.
x 360 pix.
= 453.6 Å		1.26 Å/pix.
x 360 pix.
= 453.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.26 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 4000.0 / Movie #1: 2000
Minimum - Maximum-3949.8432617200001 - 11974.921875
Average (Standard dev.)-27.769773480000001 (±1021.781372069999975)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-180-180-179
Dimensions360360360
Spacing360360360
CellA=B=C: 453.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.261.261.26
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z453.600453.600453.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-179
NX/NY/NZ360360360
MAP C/R/S213
start NC/NR/NS-180-180-179
NC/NR/NS360360360
D min/max/mean-3949.84311974.922-27.770

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Supplemental data

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Sample components

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Entire : Mammalian translation initiation 80S HCV-IRES complex with eIF5B;...

EntireName: Mammalian translation initiation 80S HCV-IRES complex with eIF5B; PRE-like state
Components
  • Sample: Mammalian translation initiation 80S HCV-IRES complex with eIF5B; PRE-like state
  • Complex: 80S ribosome
  • RNA: tRNA
  • RNA: HCV-IRES
  • Protein or peptide: eIF5B

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Supramolecule #1000: Mammalian translation initiation 80S HCV-IRES complex with eIF5B;...

SupramoleculeName: Mammalian translation initiation 80S HCV-IRES complex with eIF5B; PRE-like state
type: sample / ID: 1000 / Number unique components: 4
Molecular weightTheoretical: 4.8 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 / Tissue: reticulocyte lysate
Molecular weightTheoretical: 4.5 MDa

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Macromolecule #1: tRNA

MacromoleculeName: tRNA / type: rna / ID: 1 / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit
Molecular weightTheoretical: 2.5 KDa

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Macromolecule #3: HCV-IRES

MacromoleculeName: HCV-IRES / type: rna / ID: 3 / Classification: OTHER / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Hepatitis C virus / synonym: HCV
Molecular weightTheoretical: 162 KDa
SequenceString: GCCAGCCCCC UGAUGGGGGC GACACUCCAC CAUGAAUCAC UCCCCUGUGA GGAACUACUG UCUUCACGCA GAAAGCGUCU AGCCAUGGCG UUAGUAUGAG UGUCGUGCAG CCUCCAGGAC CCCCCCUCCC GGGAGAGCCA UAGUGGUCUG CGGAACCGGU GAGUACACCG ...String:
GCCAGCCCCC UGAUGGGGGC GACACUCCAC CAUGAAUCAC UCCCCUGUGA GGAACUACUG UCUUCACGCA GAAAGCGUCU AGCCAUGGCG UUAGUAUGAG UGUCGUGCAG CCUCCAGGAC CCCCCCUCCC GGGAGAGCCA UAGUGGUCUG CGGAACCGGU GAGUACACCG GAAUUGCCAG GACGACCGGG UCCUUUCUUG GAUAAACCCG CUCAAUGCCU GGAGAUUUGG GCGUGCCCCC GCAAGACUGC UAGCCGAGUA GUGUUGGGUC GCGAAAGGCC UUGUGGUACU GCCUGAUAGG GUGCUUGCGA GUGCCCCGGG AGGUCUCGUA GACCGUGCAC CAUGAGCACG AAUCCUAAAC CUCAAAGAAA AACCAAACGU AACACCAACC GUCGCCCACA GGACGUCAAG UUCCCGGGUG GCGGUCUAGA CGCCGAGAUC AGAAAUCCCU CUCUCGGAUC GCAUUUGGAC UUCUGCCUUC GGGCACCACG GUCGGAUCCG AAUU

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Macromolecule #2: eIF5B

MacromoleculeName: eIF5B / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Recombinant expression: No
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Tissue: reticulocyte lysate
Molecular weightTheoretical: 160 KDa
SequenceUniProtKB: Eukaryotic translation initiation factor 5B

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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

Concentration0.15 mg/mL
BufferpH: 7.6
Details: 20mM Tris-HCl, 5mM MgCl2, 100mM KCl, 0.2mM spermidine, 2mM DTT
GridDetails: Quantifoil R3-3 Cu 300 mesh with 2 nm carbon support film
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 93 K / Instrument: FEI VITROBOT MARK II / Method: blot for 2-4 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 115,000 times magnification
Detailsautomated data collection using Leginon
DateDec 22, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 22287 / Average electron dose: 20 e/Å2 / Details: Automated data collection on using Leginon / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 194805 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 115000
Sample stageSpecimen holder: Cartridge system, LN2 cooled / Specimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND3
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.9 Å / Resolution method: OTHER / Software - Name: spider, sparx / Number images used: 541570

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