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- PDB-6i3m: eIF2B:eIF2 complex, phosphorylated on eIF2 alpha serine 52. -

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Basic information

Entry
Database: PDB / ID: 6i3m
TitleeIF2B:eIF2 complex, phosphorylated on eIF2 alpha serine 52.
Components
  • (Eukaryotic translation initiation factor 2 subunit ...) x 3
  • (Translation initiation factor eIF-2B subunit ...) x 5
KeywordsTRANSLATION / Translational control eIF2 phosphorylation Integrated stress response eIF2B TRANSLATION
Function / homology
Function and homology information


negative regulation of cellular response to amino acid starvation / Recycling of eIF2:GDP / ABC-family proteins mediated transport / methionyl-initiator methionine tRNA binding / eukaryotic translation initiation factor 2B complex / eukaryotic translation initiation factor 2 complex / multi-eIF complex / eukaryotic 43S preinitiation complex / cytoplasmic translational initiation / protein-synthesizing GTPase ...negative regulation of cellular response to amino acid starvation / Recycling of eIF2:GDP / ABC-family proteins mediated transport / methionyl-initiator methionine tRNA binding / eukaryotic translation initiation factor 2B complex / eukaryotic translation initiation factor 2 complex / multi-eIF complex / eukaryotic 43S preinitiation complex / cytoplasmic translational initiation / protein-synthesizing GTPase / formation of translation preinitiation complex / formation of cytoplasmic translation initiation complex / guanyl-nucleotide exchange factor complex / eukaryotic 48S preinitiation complex / positive regulation of cellular response to amino acid starvation / positive regulation of translational fidelity / regulation of translational initiation / Formation of the ternary complex, and subsequently, the 43S complex / Translation initiation complex formation / Ribosomal scanning and start codon recognition / L13a-mediated translational silencing of Ceruloplasmin expression / enzyme regulator activity / translation initiation factor binding / translational initiation / translation initiation factor activity / guanyl-nucleotide exchange factor activity / cytoplasmic stress granule / ribosome binding / ribosome / mRNA binding / GTPase activity / GTP binding / mitochondrion / RNA binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Methylthioribose-1-phosphate isomerase, N-terminal / Translation initiation factor eIF-2B subunit alpha, N-terminal / Translation initiation factor eIF-2B subunit epsilon, N-terminal / Translation initiation factor eIF-2B subunit epsilon, W2 domain / Translation initiation factor IF2/IF5 / Translation initiation factor IF2/IF5 domain / Translation initiation factor IF2/IF5, N-terminal / Translation initiation factor IF2/IF5, zinc-binding / Domain found in IF2B/IF5 / domain present in translation initiation factor eIF2B and eIF5 ...Methylthioribose-1-phosphate isomerase, N-terminal / Translation initiation factor eIF-2B subunit alpha, N-terminal / Translation initiation factor eIF-2B subunit epsilon, N-terminal / Translation initiation factor eIF-2B subunit epsilon, W2 domain / Translation initiation factor IF2/IF5 / Translation initiation factor IF2/IF5 domain / Translation initiation factor IF2/IF5, N-terminal / Translation initiation factor IF2/IF5, zinc-binding / Domain found in IF2B/IF5 / domain present in translation initiation factor eIF2B and eIF5 / Initiation factor 2B-related / Initiation factor 2B-like, C-terminal / Initiation factor 2 subunit family / eIF4-gamma/eIF5/eIF2-epsilon / Domain at the C-termini of GCD6, eIF-2B epsilon, eIF-4 gamma and eIF-5 / W2 domain / W2 domain profile. / IF2a, S1-like domain / Translation initiation factor 2, alpha subunit / Translation initiation factor 2, alpha subunit, middle domain superfamily / Translation initiation factor 2, alpha subunit, C-terminal / Eukaryotic translation initiation factor 2 alpha subunit / Initiation factor eIF2 gamma, domain 2 / Initiation factor eIF2 gamma, GTP-binding domain / Initiation factor eIF2 gamma, C-terminal / Initiation factor eIF2 gamma, C terminal / Nucleotidyl transferase domain / Nucleotidyl transferase / NagB/RpiA transferase-like / Bacterial transferase hexapeptide (six repeats) / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Trimeric LpxA-like superfamily / S1 domain profile. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Nucleotide-diphospho-sugar transferases / Translation protein, beta-barrel domain superfamily / Armadillo-type fold / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Translation initiation factor eIF2B subunit gamma / Eukaryotic translation initiation factor 2 subunit beta / Translation initiation factor eIF2B subunit delta / Translation initiation factor eIF2B subunit alpha / Eukaryotic translation initiation factor 2 subunit alpha / Eukaryotic translation initiation factor 2 subunit gamma / Translation initiation factor eIF2B subunit epsilon / Translation initiation factor eIF2B subunit beta
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.93 Å
AuthorsAdomavicius, T. / 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
Citation
Journal: Nat Commun / Year: 2019
Title: The structural basis of translational control by eIF2 phosphorylation.
Authors: Tomas Adomavicius / Margherita Guaita / Yu Zhou / Martin D Jennings / Zakia Latif / Alan M Roseman / Graham D Pavitt /
Abstract: Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 ...Protein synthesis in eukaryotes is controlled by signals and stresses via a common pathway, called the integrated stress response (ISR). Phosphorylation of the translation initiation factor eIF2 alpha at a conserved serine residue mediates translational control at the ISR core. To provide insight into the mechanism of translational control we have determined the structures of eIF2 both in phosphorylated and unphosphorylated forms bound with its nucleotide exchange factor eIF2B by electron cryomicroscopy. The structures reveal that eIF2 undergoes large rearrangements to promote binding of eIF2α to the regulatory core of eIF2B comprised of the eIF2B alpha, beta and delta subunits. Only minor differences are observed between eIF2 and eIF2αP binding to eIF2B, suggesting that the higher affinity of eIF2αP for eIF2B drives translational control. We present a model for controlled nucleotide exchange and initiator tRNA binding to the eIF2/eIF2B complex.
#1: Journal: 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.
History
DepositionNov 6, 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
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: em_3d_fitting / Item: _em_3d_fitting.target_criteria

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Structure visualization

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  • Deposited structure unit
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Structure viewerMolecule:
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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
H: Translation initiation factor eIF-2B subunit epsilon
F: Translation initiation factor eIF-2B subunit beta
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)838,12716
Polymers838,12716
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, eIF2 and eIF2B purified separately as complexes from yeast saccharomyces cerevisiae. eIF2 complexes phosphorylated by separately purified PKR kinase in vitro. Assembly ...Evidence: gel filtration, eIF2 and eIF2B purified separately as complexes from yeast saccharomyces cerevisiae. eIF2 complexes phosphorylated by separately purified PKR kinase in vitro. Assembly generated by mixing complexes and isolating on gel filtration column.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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

#1: Protein Translation initiation factor eIF-2B subunit alpha / GCD complex subunit GCN3 / Guanine nucleotide exchange factor subunit GCN3 / Transcriptional ...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 S288C (yeast) / Gene: GCN3, AAS2, TIF221, YKR026C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P14741
#2: Protein Translation initiation factor eIF-2B subunit delta / GCD complex subunit GCD2 / Guanine nucleotide exchange factor subunit GCD2 / eIF-2B GDP-GTP ...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 S288C (yeast) / Gene: GCD2, TIF224, YGR083C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P12754
#3: Protein Translation initiation factor eIF-2B subunit beta / GCD complex subunit GCD7 / Guanine nucleotide exchange factor subunit GCD7 / eIF-2B GDP-GTP ...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 S288C (yeast) / Gene: GCD7, TIF222, YLR291C, L8003.17 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32502
#4: Protein Translation initiation factor eIF-2B subunit epsilon / GCD complex subunit GCD6 / Guanine nucleotide exchange factor subunit GCD6 / eIF-2B GDP-GTP ...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 S288C (yeast) / Gene: GCD6, TIF225, YDR211W, YD8142.12, YD8142B.03 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P32501
#5: Protein Translation initiation factor eIF-2B subunit gamma / GCD complex subunit GCD1 / Guanine nucleotide exchange factor subunit GCD1 / eIF-2B GDP-GTP ...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 S288C (yeast) / Gene: GCD1, TIF223, TRA3, YOR260W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P09032

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

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


Mass: 34843.633 Da / Num. of mol.: 2 / Mutation: serine 52 to SEP
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Gene: SUI2, TIF211, YJR007W, J1429 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P20459
#7: Protein 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 S288C (yeast) / Gene: SUI3, TIF212, YPL237W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P09064
#8: Protein 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 S288C (yeast) / Gene: GCD11, TIF213, YER025W / Production host: Saccharomyces cerevisiae (brewer'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: all / Source: RECOMBINANT
Molecular weightValue: 0.838 MDa / Experimental value: YES
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
Details: Solutions for sample preparation were made fresh, filter sterilized, and degassed.
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium chlorideKCl1
220 mMTris1
31 mMTCEP1
SpecimenConc.: 0.25 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Ultra-thin carbon support film, 3nm - on lacey carbon grids from Agar Scientific were used for 35 degree tilted data collection. For zero tilt data collection, 200 mesh Au Quantifoil, R2/2 grids were used.
Grid 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 2 seconds 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 zero and 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: 4000 nm / Nominal defocus min: 1500 nm / Calibrated defocus min: 1000 nm / Calibrated defocus max: 5500 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 12 sec. / Electron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 4533
Details: 2 separate data collections for zero degree (2255 images) and tilted (2278 images) specimen. Both on Titan Krios
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 48 / Used frames/image: 2-48

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Processing

EM software
IDNameVersionCategoryFitting-IDDetails
1RELION2.01particle selection
3EPUimage acquisition
5GctfCTF correction
8MODELLERmodel fitting1
9UCSF Chimeramodel fitting1
10Cootmodel fitting1
14RELION2.1initial Euler assignment
15RELION2.1final Euler assignment
17RELION2.1classification
18RELION2.13D reconstruction
20PHENIXmodel refinement1Core region and C-term of eIF2 alpha were modelled directly into the map, using Coot and refined in Phenix
21UCSF 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: 655000
Details: Initially, approximately 3000 particles were picked manually and used for 2D classification. Then 4 best looking 2D class average images were used as references for automated particle ...Details: Initially, approximately 3000 particles were picked manually and used for 2D classification. Then 4 best looking 2D class average images were used as references for automated particle picking in RELION. For the zero tilt data collection, 406000 particles were picked. For the 35 degree tilt data collection, 249000 particles were picked
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.93 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 64541 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceTarget criteriaDetails
1FLEXIBLE FITREALcorrelation coeficientModeller was used to build homology model of S. cerevisiae eIF2B structure based on S. pombe crystal structure. Subunits of the homology model, along with eIF2 alpha domains 1 and 2, were then rigid body fitted into our map using UCSF Chimera. The model then was refined using phenix and manually adjusted in Coot.
2RIGID BODY FITREALCross-correlation coefficienteIF2 alpha domain 3, eIF2 gamma, and eIF2 beta (3JAP) were rigid body fitted into our map using Chimera.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
15B04

5b04

1
23JAP

3jap

j13-174
33JAP

3jap

j2182-265
43JAP

3jap

k2
53JAP

3jap

l2127-143

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