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- PDB-6ezo: Eukaryotic initiation factor EIF2B in complex with ISRIB -

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Basic information

Entry
Database: PDB / ID: 6ezo
TitleEukaryotic initiation factor EIF2B in complex with ISRIB
Components
  • (Translation initiation factor eIF-2B subunit ...) x 4
  • Human eukaryotic initiation factor EIF2B epsilon subunits
KeywordsMEMBRANE PROTEIN / GEF / Complex / ISRIB
Function / homology
Function and homology information


eukaryotic translation initiation factor 2B complex / Recycling of eIF2:GDP / cytoplasmic translational initiation / guanyl-nucleotide exchange factor complex / oligodendrocyte development / astrocyte development / astrocyte differentiation / regulation of translational initiation / positive regulation of translational initiation / response to glucose ...eukaryotic translation initiation factor 2B complex / Recycling of eIF2:GDP / cytoplasmic translational initiation / guanyl-nucleotide exchange factor complex / oligodendrocyte development / astrocyte development / astrocyte differentiation / regulation of translational initiation / positive regulation of translational initiation / response to glucose / ovarian follicle development / translational initiation / translation initiation factor binding / translation initiation factor activity / myelination / response to endoplasmic reticulum stress / guanyl-nucleotide exchange factor activity / central nervous system development / hippocampus development / response to peptide hormone / regulation of translation / T cell receptor signaling pathway / response to heat / positive regulation of apoptotic process / GTP binding / ATP binding / membrane / identical protein binding / nucleus / plasma membrane / cytosol / cytoplasm
Similarity search - Function
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 / 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. ...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 / 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. / Nucleotidyl transferase domain / Nucleotidyl transferase / NagB/RpiA transferase-like / Bacterial transferase hexapeptide (six repeats) / Trimeric LpxA-like superfamily / Nucleotide-diphospho-sugar transferases / Armadillo-type fold
Similarity search - Domain/homology
Chem-C7B / Translation initiation factor eIF2B subunit beta / Translation initiation factor eIF2B subunit epsilon / Translation initiation factor eIF2B subunit alpha / Translation initiation factor eIF2B subunit gamma / Translation initiation factor eIF2B subunit delta
Similarity search - Component
Biological speciesHomo sapiens (human)
Homo Sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsFaille, A. / Weis, F. / Zyryanova, A. / Warren, A.J. / Ron, D.
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome TrustWT200848/Z/16/Z United Kingdom
Medical Research Council (United Kingdom)RG67621 United Kingdom
CitationJournal: Science / Year: 2018
Title: Binding of ISRIB reveals a regulatory site in the nucleotide exchange factor eIF2B.
Authors: Alisa F Zyryanova / Félix Weis / Alexandre Faille / Akeel Abo Alard / Ana Crespillo-Casado / Yusuke Sekine / Heather P Harding / Felicity Allen / Leopold Parts / Christophe Fromont / Peter ...Authors: Alisa F Zyryanova / Félix Weis / Alexandre Faille / Akeel Abo Alard / Ana Crespillo-Casado / Yusuke Sekine / Heather P Harding / Felicity Allen / Leopold Parts / Christophe Fromont / Peter M Fischer / Alan J Warren / David Ron /
Abstract: The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of ...The integrated stress response (ISR) is a conserved translational and transcriptional program affecting metabolism, memory, and immunity. The ISR is mediated by stress-induced phosphorylation of eukaryotic translation initiation factor 2α (eIF2α) that attenuates the guanine nucleotide exchange factor eIF2B. A chemical inhibitor of the ISR, ISRIB, reverses the attenuation of eIF2B by phosphorylated eIF2α, protecting mice from neurodegeneration and traumatic brain injury. We describe a 4.1-angstrom-resolution cryo-electron microscopy structure of human eIF2B with an ISRIB molecule bound at the interface between the β and δ regulatory subunits. Mutagenesis of residues lining this pocket altered the hierarchical cellular response to ISRIB analogs in vivo and ISRIB binding in vitro. Our findings point to a site in eIF2B that can be exploited by ISRIB to regulate translation.
History
DepositionNov 16, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 28, 2018Provider: repository / Type: Initial release
Revision 1.1Apr 4, 2018Group: Author supporting evidence / Data collection / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Apr 11, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.3Oct 17, 2018Group: Data collection / Refinement description / Category: refine
Revision 1.4Dec 11, 2019Group: Other / Structure summary / Category: atom_sites / chem_comp
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][2] / _atom_sites.fract_transf_matrix[3][3] / _chem_comp.pdbx_synonyms
Revision 1.5May 15, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Translation initiation factor eIF-2B subunit alpha
B: Translation initiation factor eIF-2B subunit alpha
C: Translation initiation factor eIF-2B subunit beta
D: Translation initiation factor eIF-2B subunit beta
E: Translation initiation factor eIF-2B subunit gamma
F: Translation initiation factor eIF-2B subunit gamma
G: Translation initiation factor eIF-2B subunit delta
H: Translation initiation factor eIF-2B subunit delta
I: Human eukaryotic initiation factor EIF2B epsilon subunits
J: Human eukaryotic initiation factor EIF2B epsilon subunits
hetero molecules


Theoretical massNumber of molelcules
Total (without water)528,29811
Polymers527,84710
Non-polymers4511
Water0
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19070 Å2
ΔGint-156 kcal/mol
Surface area171760 Å2
MethodPISA

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Components

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Translation initiation factor eIF-2B subunit ... , 4 types, 8 molecules ABCDEFGH

#1: Protein Translation initiation factor eIF-2B subunit alpha / eIF-2B GDP-GTP exchange factor subunit alpha


Mass: 33754.148 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: epithelial / Tissue: cervix / References: UniProt: Q14232
#2: Protein Translation initiation factor eIF-2B subunit beta / S20I15 / S20III15 / eIF-2B GDP-GTP exchange factor subunit beta


Mass: 41758.242 Da / Num. of mol.: 2
Mutation: 3xFLAG inserted at position +4 of the protein sequence
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Tissue: cervix / Cell line: epithelial / Gene: EIF2B2, EIF2BB / Plasmid: HeLa-2C2 / Cell line (production host): HeLa / Production host: Homo sapiens (human) / References: UniProt: P49770
#3: Protein Translation initiation factor eIF-2B subunit gamma / eIF-2B GDP-GTP exchange factor subunit gamma


Mass: 50304.230 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: epithelial / Tissue: cervix / References: UniProt: Q9NR50
#4: Protein Translation initiation factor eIF-2B subunit delta / eIF-2B GDP-GTP exchange factor subunit delta


Mass: 57640.168 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo sapiens (human) / Cell line: epithelial / Tissue: cervix / References: UniProt: Q9UI10

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Protein / Non-polymers , 2 types, 3 molecules IJ

#5: Protein Human eukaryotic initiation factor EIF2B epsilon subunits


Mass: 80466.609 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Homo Sapiens (human) / Cell line: epithelial / Tissue: cervix / References: UniProt: Q13144
#6: Chemical ChemComp-C7B / 2-(4-chloranylphenoxy)-~{N}-[4-[2-(4-chloranylphenoxy)ethanoylamino]cyclohexyl]ethanamide / ISRIB / ISRIB


Mass: 451.343 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C22H24Cl2N2O4

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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

Component
IDNameTypeEntity IDParent-IDSource
1Eukaryotic initiation factor EIF2B in complex with ISRIBCOMPLEX#1-#50MULTIPLE SOURCES
3ISRIBCOMPLEX#21RECOMBINANT
2Eukaryotic translation initiation factor EIF2BCOMPLEX#1, #3-#51NATURAL
Molecular weightValue: 0.521 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Homo sapiens (human)9606
33Homo sapiens (human)9606
Source (recombinant)Organism: Homo sapiens (human) / Cell: HeLa-2C2
Buffer solutionpH: 7.4
SpecimenConc.: 2.65 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 80000 X / Nominal defocus max: 3500 nm / Nominal defocus min: 2500 nm / Cs: 2.7 mm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.5 sec. / Electron dose: 30 e/Å2 / Detector mode: INTEGRATING / Film or detector model: FEI FALCON II (4k x 4k) / Num. of real images: 765
Image scansMovie frames/image: 25

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Processing

SoftwareName: REFMAC / Version: 5.8.0158 / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
4GctfCTF correction
9REFMACmodel refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 237486
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41750 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: RECIPROCAL
RefinementResolution: 4.1→420 Å / Cor.coef. Fo:Fc: 0.847 / SU B: 21.345 / SU ML: 0.262 / ESU R: 0.15
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
RfactorNum. reflection% reflection
Rwork0.39443 --
obs0.39443 2251317 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 71.551 Å2
Baniso -1Baniso -2Baniso -3
1--9.3 Å2-1.35 Å20.03 Å2
2--16.47 Å2-0.1 Å2
3----7.16 Å2
Refinement stepCycle: 1 / Total: 18676
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0070.01918957
ELECTRON MICROSCOPYr_bond_other_d
ELECTRON MICROSCOPYr_angle_refined_deg1.3481.94126336
ELECTRON MICROSCOPYr_angle_other_deg
ELECTRON MICROSCOPYr_dihedral_angle_1_deg8.88253339
ELECTRON MICROSCOPYr_dihedral_angle_2_deg36.25120.094213
ELECTRON MICROSCOPYr_dihedral_angle_3_deg15.04615612
ELECTRON MICROSCOPYr_dihedral_angle_4_deg10.2221520
ELECTRON MICROSCOPYr_chiral_restr0.0840.23344
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.02115266
ELECTRON MICROSCOPYr_gen_planes_other
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it3.94210.33413432
ELECTRON MICROSCOPYr_mcbond_other
ELECTRON MICROSCOPYr_mcangle_it7.36215.48516743
ELECTRON MICROSCOPYr_mcangle_other
ELECTRON MICROSCOPYr_scbond_it2.6359.1945525
ELECTRON MICROSCOPYr_scbond_other
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other
ELECTRON MICROSCOPYr_long_range_B_refined17.13664322
ELECTRON MICROSCOPYr_long_range_B_other
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
LS refinement shellResolution: 4.1→4.207 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rwork0.626 167076 -
Rfree-0 -
obs--100 %

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