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- PDB-6i7t: eIF2B:eIF2 complex -

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Basic information

Entry
Database: PDB / ID: 6i7t
TitleeIF2B:eIF2 complex
Components
  • (Eukaryotic translation initiation factor 2 subunit ...) x 3
  • (Translation initiation factor eIF-2B subunit ...) x 5
KeywordsTRANSLATION / Translational control eIF2 Integrated stress response eIF2B TRANSLATION
Function / homology
Function and homology information


L13a-mediated translational silencing of Ceruloplasmin expression / ABC-family proteins mediated transport / Formation of the ternary complex, and subsequently, the 43S complex / Ribosomal scanning and start codon recognition / Recycling of eIF2:GDP / negative regulation of cellular response to amino acid starvation / guanyl-nucleotide exchange factor complex / eukaryotic translation initiation factor 2 complex / eukaryotic translation initiation factor 2B complex / multi-eIF complex ...L13a-mediated translational silencing of Ceruloplasmin expression / ABC-family proteins mediated transport / Formation of the ternary complex, and subsequently, the 43S complex / Ribosomal scanning and start codon recognition / Recycling of eIF2:GDP / negative regulation of cellular response to amino acid starvation / guanyl-nucleotide exchange factor complex / eukaryotic translation initiation factor 2 complex / eukaryotic translation initiation factor 2B complex / multi-eIF complex / eukaryotic 43S preinitiation complex / eukaryotic 48S preinitiation complex / formation of cytoplasmic translation initiation complex / formation of translation preinitiation complex / regulation of translational initiation / positive regulation of cellular response to amino acid starvation / nucleotidyltransferase activity / positive regulation of translational fidelity / enzyme regulator activity / translational initiation / translation initiation factor binding / guanyl-nucleotide exchange factor activity / translation initiation factor activity / cytoplasmic stress granule / ribosome binding / ribosome / GTPase activity / mRNA binding / GTP binding / mitochondrion / metal ion binding / cytosol / cytoplasm
Nucleic acid-binding, OB-fold / Trimeric LpxA-like superfamily / Initiation factor 2B-related / Transcription factor, GTP-binding domain / Hexapeptide repeat / Translation initiation factor IF2/IF5 / S1 domain / W2 domain / Translation elongation factor EFTu-like, domain 2 / Nucleotidyl transferase domain ...Nucleic acid-binding, OB-fold / Trimeric LpxA-like superfamily / Initiation factor 2B-related / Transcription factor, GTP-binding domain / Hexapeptide repeat / Translation initiation factor IF2/IF5 / S1 domain / W2 domain / Translation elongation factor EFTu-like, domain 2 / Nucleotidyl transferase domain / Translation protein, beta-barrel domain superfamily / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Translation initiation factor 2, alpha subunit / Translation initiation factor eIF-2B subunit epsilon, N-terminal / Translation initiation factor 2, gamma subunit, C-terminal / MIF4G-like domain superfamily / Armadillo-type fold / Translation initiation factor IF2/IF5, N-terminal / Translation initiation factor IF2/IF5, zinc-binding / RNA-binding domain, S1 / Translation initiation factor 2, alpha subunit, middle domain superfamily / Translation initiation factor 2, alpha subunit, C-terminal / Methylthioribose-1-phosphate isomerase, N-terminal / P-loop containing nucleoside triphosphate hydrolase / NagB/RpiA transferase-like / Nucleotide-diphospho-sugar transferases / Elongation factor Tu GTP binding domain / eIF4-gamma/eIF5/eIF2-epsilon / Initiation factor eIF2 gamma, C terminal / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Bacterial transferase hexapeptide (six repeats) / Eukaryotic translation initiation factor 2 alpha subunit / Elongation factor Tu domain 2 / W2 domain profile. / Domain found in IF2B/IF5 / Initiation factor 2 subunit family / S1 RNA binding domain / Nucleotidyl transferase / S1 domain profile.
Translation initiation factor eIF-2B subunit gamma / Eukaryotic translation initiation factor 2 subunit beta / Translation initiation factor eIF-2B subunit delta / Translation initiation factor eIF-2B subunit alpha / Eukaryotic translation initiation factor 2 subunit alpha / Eukaryotic translation initiation factor 2 subunit gamma / Translation initiation factor eIF-2B subunit epsilon / Translation initiation factor eIF-2B subunit beta
Biological speciesSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.61 Å
AuthorsAdomavicius, T. / Guaita, M. / Roseman, A.M. / Pavitt, G.D.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/L020157/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/N014049/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/M006565/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/M011208/1 United Kingdom
CitationJournal: Elife / Year: 2017
Title: Fail-safe control of translation initiation by dissociation of eIF2α phosphorylated ternary complexes.
Authors: Martin D Jennings / Christopher J Kershaw / Tomas Adomavicius / Graham D Pavitt /
Abstract: Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and ...Phosphorylation of eIF2α controls translation initiation by restricting the levels of active eIF2-GTP/Met-tRNAi ternary complexes (TC). This modulates the expression of all eukaryotic mRNAs and contributes to the cellular integrated stress response. Key to controlling the activity of eIF2 are translation factors eIF2B and eIF5, thought to primarily function with eIF2-GDP and TC respectively. Using a steady-state kinetics approach with purified proteins we demonstrate that eIF2B binds to eIF2 with equal affinity irrespective of the presence or absence of competing guanine nucleotides. We show that eIF2B can compete with Met-tRNAi for eIF2-GTP and can destabilize TC. When TC is formed with unphosphorylated eIF2, eIF5 can out-compete eIF2B to stabilize TC/eIF5 complexes. However when TC/eIF5 is formed with phosphorylated eIF2, eIF2B outcompetes eIF5 and destabilizes TC. These data uncover competition between eIF2B and eIF5 for TC and identify that phosphorylated eIF2-GTP translation initiation intermediate complexes can be inhibited by eIF2B.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 17, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 22, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 19, 2019Group: Data collection / Database references
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_database_proc
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-4428
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Translation initiation factor eIF-2B subunit alpha
C: Translation initiation factor eIF-2B subunit delta
D: Translation initiation factor eIF-2B subunit delta
E: Translation initiation factor eIF-2B subunit beta
G: Translation initiation factor eIF-2B subunit epsilon
I: Translation initiation factor eIF-2B subunit gamma
J: Translation initiation factor eIF-2B subunit gamma
B: Translation initiation factor eIF-2B subunit alpha
F: Translation initiation factor eIF-2B subunit beta
H: Translation initiation factor eIF-2B subunit epsilon
K: Eukaryotic translation initiation factor 2 subunit alpha
M: Eukaryotic translation initiation factor 2 subunit beta
O: Eukaryotic translation initiation factor 2 subunit gamma
L: Eukaryotic translation initiation factor 2 subunit alpha
N: Eukaryotic translation initiation factor 2 subunit beta
P: Eukaryotic translation initiation factor 2 subunit gamma


Theoretical massNumber of molelcules
Total (without water)837,96716
Polymers837,96716
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, eIF2 and eIF2B were purified separately as complexes from yeast saccharomyces cerevisiae. Assembly was generated by mixing complexes, and isolated on a gel filtration column.
  • Download structure data
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area48790 Å2
ΔGint-235 kcal/mol
Surface area209550 Å2
MethodPISA

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Components

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Translation initiation factor eIF-2B subunit ... , 5 types, 10 molecules ABCDEFGHIJ

#1: Protein/peptide Translation initiation factor eIF-2B subunit alpha / GCD complex subunit GCN3 / Guanine nucleotide exchange factor subunit GCN3 / Transcriptional activator GCN3 / eIF-2B GDP-GTP exchange factor subunit alpha


Mass: 34062.027 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: GCN3, AAS2, TIF221, YKR026C / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P14741
#2: Protein/peptide Translation initiation factor eIF-2B subunit delta / GCD complex subunit GCD2 / Guanine nucleotide exchange factor subunit GCD2 / eIF-2B GDP-GTP exchange factor subunit delta


Mass: 70945.195 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: GCD2, TIF224, YGR083C / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P12754
#3: Protein/peptide Translation initiation factor eIF-2B subunit beta / GCD complex subunit GCD7 / Guanine nucleotide exchange factor subunit GCD7 / eIF-2B GDP-GTP exchange factor subunit beta


Mass: 42621.441 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: GCD7, TIF222, YLR291C, L8003.17 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32502
#4: Protein/peptide Translation initiation factor eIF-2B subunit epsilon / GCD complex subunit GCD6 / Guanine nucleotide exchange factor subunit GCD6 / eIF-2B GDP-GTP exchange factor subunit epsilon


Mass: 81249.062 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: GCD6, TIF225, YDR211W, YD8142.12, YD8142B.03 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32501
#5: Protein/peptide Translation initiation factor eIF-2B subunit gamma / GCD complex subunit GCD1 / Guanine nucleotide exchange factor subunit GCD1 / eIF-2B GDP-GTP exchange factor subunit gamma


Mass: 65768.320 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: GCD1, TIF223, TRA3, YOR260W / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P09032

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Eukaryotic translation initiation factor 2 subunit ... , 3 types, 6 molecules KLMNOP

#6: Protein/peptide Eukaryotic translation initiation factor 2 subunit alpha / eIF-2-alpha


Mass: 34763.652 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: SUI2, TIF211, YJR007W, J1429 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P20459
#7: Protein/peptide Eukaryotic translation initiation factor 2 subunit beta / eIF-2-beta


Mass: 31631.309 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: SUI3, TIF212, YPL237W / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P09064
#8: Protein/peptide Eukaryotic translation initiation factor 2 subunit gamma / eIF-2-gamma


Mass: 57942.699 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Gene: GCD11, TIF213, YER025W / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: P32481

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of translation initiation factors eIF2 and eIF2B
Type: COMPLEX / Entity ID: 1, 2, 3, 4, 5, 6, 7, 8 / Source: RECOMBINANT
Molecular weightValue: 0.838 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (baker's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionpH: 7.5
Details: Solutions for sample preparation were made fresh, filter sterilized, and degassed.
Buffer component

Buffer-ID: 1

IDConc.NameFormula
1100 mMpotassium chlorideKCl
220 mMTris
31 mMTCEP
SpecimenConc.: 0.28 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 294 K / Details: Blot for 1 second before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Details: Tilt at 35 degrees.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 37313 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 261 nm / Calibrated defocus max: 4601 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2154
Details: Images were taken with 40, 60 or 100 frames in total. The first 2 frames were discarded in motioncorr2. The stage was tilted by 35 degrees.
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µns / Width: 3838 / Height: 3710 / Movie frames/image: 100 / Used frames/image: 2-100

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Processing

EM software
IDNameVersionCategoryFitting-IDDetails
1RELION2.1particle selection
3EPUimage acquisition
5GctfCTF correction
8UCSF Chimeramodel fitting1
9Cootmodel fitting1
13RELION2.1initial Euler assignment
14RELION2.1final Euler assignment
16RELION2.1classification
17RELION2.13D reconstruction
19PHENIXmodel refinement1Core region and C-term of eIF2 alpha were modelled directly into the map, using Coot and refined in Phenix
20UCSF Chimeramodel fitting2cross-correlation at 15 A
CTF correctionDetails: CTF determination was performed per micrograph initially. After particle picking and initial reconstruction, per particle CTF determination was performed.
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 114390
Details: 2024 images were selected for particle picking. Initially, 3090 particles were picked manually and used for 2D classification into 20 classes. The best 8 classes (containing 2162 particles) were selected and used as references for automated particle picking in RELION. Particles on the lacey carbon, not in holes, were manually deleted in RELION, before extraction and further iterative cleaning by classification.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 23274 / Symmetry type: POINT
Atomic model building

Space: REAL

IDProtocolTarget criteriaDetails
1FLEXIBLE FITcorrelation coeficientOur model of eIF2aP:eIF2B (PDB code:6I3M) was fitted and then iteratively refined using phenix and manually adjusted in Coot.
2RIGID BODY FITCorrelation CoefficienteIF2 alpha domain 3, eIF2 gamma, and eIF2 beta (3JAP) were rigid body fitted into our map, Fourier filtered 15A resolution, using UCSF_Chimera.
Atomic model building
IDPDB-IDPdb chain-ID3D fitting-IDPdb chain residue range
16I3MA1
26I3MB1
36I3MC1
46I3MD1
56I3ME1
66I3MF1
76I3MG1
86I3MH1
96I3MI1
106I3MJ1
116I3MK13-174
126I3ML13-174
133JAPj2182-265
143JAPk2
153JAPl2127-143

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