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- EMDB-4404: eIF2B:eIF2 complex, phosphorylated on eIF2 alpha serine 52. -

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Basic information

Entry
Database: EMDB / ID: EMD-4404
TitleeIF2B:eIF2 complex, phosphorylated on eIF2 alpha serine 52.
Map data
SampleComplex of translation initiation factors eIF2 and eIF2B
  • (Translation initiation factor eIF-2B subunit ...) x 5
  • (Eukaryotic translation initiation factor 2 subunit ...) x 3
Function / homology
Function and homology information


Recycling of eIF2:GDP / Ribosomal scanning and start codon recognition / Formation of the ternary complex, and subsequently, the 43S complex / ABC-family proteins mediated transport / L13a-mediated translational silencing of Ceruloplasmin expression / 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 ...Recycling of eIF2:GDP / Ribosomal scanning and start codon recognition / Formation of the ternary complex, and subsequently, the 43S complex / ABC-family proteins mediated transport / L13a-mediated translational silencing of Ceruloplasmin expression / 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 translation preinitiation complex / regulation of translational initiation / positive regulation of cellular response to amino acid starvation / nucleotidyltransferase activity / enzyme regulator activity / translational initiation / guanyl-nucleotide exchange factor activity / translation initiation factor binding / positive regulation of translational fidelity / 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 / Translation initiation factor 2, alpha subunit / 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 ...Nucleic acid-binding, OB-fold / Translation initiation factor 2, alpha subunit / 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 / Trimeric LpxA-like superfamily / NagB/RpiA transferase-like / Elongation factor Tu GTP binding domain / 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 / Initiation factor 2B-related / Nucleotide-diphospho-sugar transferases / Translation initiation factor eIF-2B subunit epsilon, N-terminal / Bacterial transferase hexapeptide (six repeats) / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / W2 domain profile. / S1 domain profile. / Initiation factor eIF2 gamma, C terminal / Eukaryotic translation initiation factor 2 alpha subunit / Elongation factor Tu domain 2 / eIF4-gamma/eIF5/eIF2-epsilon / Domain found in IF2B/IF5 / Initiation factor 2 subunit family / S1 RNA binding domain / Nucleotidyl transferase
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 (strain ATCC 204508 / S288c) (yeast) / Saccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.93 Å
AuthorsAdomavicius T / Roseman AM / Pavitt GD
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.
Validation ReportPDB-ID: 6i3m

SummaryFull reportAbout validation report
DateDeposition: Nov 6, 2018 / Header (metadata) release: May 8, 2019 / Map release: May 22, 2019 / Update: May 22, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.055
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6i3m
  • Surface level: 0.055
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_4404.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 320 pix.
= 428.8 Å
1.34 Å/pix.
x 320 pix.
= 428.8 Å
1.34 Å/pix.
x 320 pix.
= 428.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.055 / Movie #1: 0.055
Minimum - Maximum-0.19225763 - 0.532957
Average (Standard dev.)0.0010794027 (±0.014409717)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 428.80002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z428.800428.800428.800
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ513513513
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.1810.3040.000

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Supplemental data

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Mask #1

Fileemd_4404_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Complex of translation initiation factors eIF2 and eIF2B

EntireName: Complex of translation initiation factors eIF2 and eIF2B
Number of components: 9

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Component #1: protein, Complex of translation initiation factors eIF2 and eIF2B

ProteinName: Complex of translation initiation factors eIF2 and eIF2B
Recombinant expression: No
MassExperimental: 838 kDa
SourceSpecies: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, Translation initiation factor eIF-2B subunit alpha

ProteinName: Translation initiation factor eIF-2B subunit alpha / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 34.062027 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: protein, Translation initiation factor eIF-2B subunit delta

ProteinName: Translation initiation factor eIF-2B subunit delta / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 70.945195 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #4: protein, Translation initiation factor eIF-2B subunit beta

ProteinName: Translation initiation factor eIF-2B subunit beta / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 42.621441 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #5: protein, Translation initiation factor eIF-2B subunit epsilon

ProteinName: Translation initiation factor eIF-2B subunit epsilon / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 81.249062 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #6: protein, Translation initiation factor eIF-2B subunit gamma

ProteinName: Translation initiation factor eIF-2B subunit gamma / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 65.76832 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #7: protein, Eukaryotic translation initiation factor 2 subunit alpha

ProteinName: Eukaryotic translation initiation factor 2 subunit alpha
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 34.843633 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #8: protein, Eukaryotic translation initiation factor 2 subunit beta

ProteinName: Eukaryotic translation initiation factor 2 subunit beta
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 31.631309 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #9: protein, Eukaryotic translation initiation factor 2 subunit gamma

ProteinName: Eukaryotic translation initiation factor 2 subunit gamma
Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 57.942699 kDa
SourceSpecies: Saccharomyces cerevisiae S288C (yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 0.25 mg/mL
Buffer solution: Solutions for sample preparation were made fresh, filter sterilized, and degassed.
pH: 7.5
Support filmUltra-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.
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temperature: 294 K / Humidity: 100 % / Details: Blot for 2 seconds before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS / Details: Tilt at zero and 35 degrees.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 40 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 37313.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1500.0 - 4000.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 QUANTUM (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 4533 / Sampling size: 5 µm
Details: 2 separate data collections for zero degree (2255 images) and tilted (2278 images) specimen. Both on Titan Krios

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 64541
3D reconstructionSoftware: RELION
CTF correction: CTF determination was performed per micrograph initially. After particle picking and initial reconstruction, per particle CTF determination was performed.
Resolution: 3.93 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: correlation coeficient / Refinement space: REAL
Details: Modeller 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.
Input PDB model: 5B04, 3JAP
Chain ID: j
Modeling #2Refinement protocol: rigid body / Target criteria: Correlation Coefficient / Refinement space: REAL
Details: eIF2 alpha domain 3, eIF2 gamma, and eIF2 beta (3JAP) were rigid body fitted into our map using Chimera.
Input PDB model: 3JAP, 3JAP, 3JAP
Chain ID: j, k, l
Output model

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