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- PDB-8huj: Cryo-EM structure of the J-K-St region of EMCV IRES in complex wi... -

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Basic information

Entry
Database: PDB / ID: 8huj
TitleCryo-EM structure of the J-K-St region of EMCV IRES in complex with eIF4G-HEAT1 and eIF4A
Components
  • Eukaryotic initiation factor 4A-I
  • Eukaryotic translation initiation factor 4 gamma 1
  • IRES RNA (J-K-St)
KeywordsTRANSLATION / Translation initiation factors / RNA binding protein
Function / homology
Function and homology information


positive regulation of eukaryotic translation initiation factor 4F complex assembly / positive regulation of mRNA cap binding / positive regulation of translation in response to endoplasmic reticulum stress / cap-dependent translational initiation / macromolecule biosynthetic process / Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S / eukaryotic initiation factor 4E binding / regulation of cellular response to stress / RNA cap binding / eukaryotic translation initiation factor 4F complex ...positive regulation of eukaryotic translation initiation factor 4F complex assembly / positive regulation of mRNA cap binding / positive regulation of translation in response to endoplasmic reticulum stress / cap-dependent translational initiation / macromolecule biosynthetic process / Activation of the mRNA upon binding of the cap-binding complex and eIFs, and subsequent binding to 43S / eukaryotic initiation factor 4E binding / regulation of cellular response to stress / RNA cap binding / eukaryotic translation initiation factor 4F complex / Z-decay: degradation of maternal mRNAs by zygotically expressed factors / cytoplasmic translational initiation / translation factor activity, RNA binding / Deadenylation of mRNA / miRNA-mediated gene silencing by inhibition of translation / M-decay: degradation of maternal mRNAs by maternally stored factors / positive regulation of protein localization to cell periphery / regulation of translational initiation / Ribosomal scanning and start codon recognition / Translation initiation complex formation / negative regulation of peptidyl-threonine phosphorylation / mTORC1-mediated signalling / cellular response to nutrient levels / regulation of presynapse assembly / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / GTP hydrolysis and joining of the 60S ribosomal subunit / L13a-mediated translational silencing of Ceruloplasmin expression / positive regulation of G1/S transition of mitotic cell cycle / behavioral fear response / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / translation initiation factor binding / energy homeostasis / translational initiation / positive regulation of neuron differentiation / positive regulation of protein metabolic process / translation initiation factor activity / negative regulation of autophagy / helicase activity / AUF1 (hnRNP D0) binds and destabilizes mRNA / lung development / ISG15 antiviral mechanism / neuron differentiation / Regulation of expression of SLITs and ROBOs / cytoplasmic stress granule / double-stranded RNA binding / positive regulation of peptidyl-serine phosphorylation / positive regulation of cell growth / postsynapse / response to ethanol / RNA helicase activity / molecular adaptor activity / RNA helicase / ribosome / translation / mRNA binding / ATP hydrolysis activity / RNA binding / extracellular exosome / ATP binding / membrane / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
ATP-dependent RNA helicase eIF4A, DEAD-box helicase domain / Initiation factor 4G / Initiation factor eIF-4 gamma, MA3 / MA3 domain / MI domain profile. / Domain in DAP-5, eIF4G, MA-3 and other proteins. / 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. ...ATP-dependent RNA helicase eIF4A, DEAD-box helicase domain / Initiation factor 4G / Initiation factor eIF-4 gamma, MA3 / MA3 domain / MI domain profile. / Domain in DAP-5, eIF4G, MA-3 and other proteins. / 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. / MIF4G domain / Middle domain of eukaryotic initiation factor 4G (eIF4G) / MIF4G-like, type 3 / DEAD-box subfamily ATP-dependent helicases signature. / ATP-dependent RNA helicase DEAD-box, conserved site / RNA helicase, DEAD-box type, Q motif / DEAD-box RNA helicase Q motif profile. / DEAD/DEAH box helicase / DEAD/DEAH box helicase domain / Helicase conserved C-terminal domain / helicase superfamily c-terminal domain / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Armadillo-type fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / Eukaryotic initiation factor 4A-I / Eukaryotic translation initiation factor 4 gamma 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Encephalomyocarditis virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsSuzuki, H. / Fujiyoshi, Y. / Imai, S. / Shimada, I.
Funding support Japan, 2items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)21ae0121028h0001 Japan
Japan Society for the Promotion of Science (JSPS)20H00451 Japan
CitationJournal: Nat Commun / Year: 2023
Title: Dynamically regulated two-site interaction of viral RNA to capture host translation initiation factor.
Authors: Shunsuke Imai / Hiroshi Suzuki / Yoshinori Fujiyoshi / Ichio Shimada /
Abstract: Many RNA viruses employ internal ribosome entry sites (IRESs) in their genomic RNA to commandeer the host's translational machinery for replication. The IRES from encephalomyocarditis virus (EMCV) ...Many RNA viruses employ internal ribosome entry sites (IRESs) in their genomic RNA to commandeer the host's translational machinery for replication. The IRES from encephalomyocarditis virus (EMCV) interacts with eukaryotic translation initiation factor 4 G (eIF4G), recruiting the ribosomal subunit for translation. Here, we analyze the three-dimensional structure of the complex composed of EMCV IRES, the HEAT1 domain fragment of eIF4G, and eIF4A, by cryo-electron microscopy. Two distinct eIF4G-interacting domains on the IRES are identified, and complex formation changes the angle therebetween. Further, we explore the dynamics of these domains by using solution NMR spectroscopy, revealing conformational equilibria in the microsecond to millisecond timescale. In the lowly-populated conformations, the base-pairing register of one domain is shifted with the structural transition of the three-way junction, as in the complex structure. Our study provides insights into the viral RNA's sophisticated strategy for optimal docking to hijack the host protein.
History
DepositionDec 24, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 2, 2023Provider: repository / Type: Initial release
Revision 1.1Sep 13, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Eukaryotic initiation factor 4A-I
B: Eukaryotic translation initiation factor 4 gamma 1
C: IRES RNA (J-K-St)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)115,3586
Polymers115,2853
Non-polymers733
Water181
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Eukaryotic initiation factor 4A-I / eIF-4A-I / eIF4A-I / ATP-dependent RNA helicase eIF4A-1


Mass: 48659.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EIF4A1, DDX2A, EIF4A / Production host: Escherichia coli (E. coli) / References: UniProt: P60842, RNA helicase
#2: Protein Eukaryotic translation initiation factor 4 gamma 1 / / eIF-4-gamma 1 / eIF-4G 1 / eIF-4G1 / p220


Mass: 31786.811 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: EIF4G1, EIF4F, EIF4G, EIF4GI / Production host: Escherichia coli (E. coli) / References: UniProt: Q04637
#3: RNA chain IRES RNA (J-K-St)


Mass: 34838.609 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Encephalomyocarditis virus / References: GenBank: NC_001479.1
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Mg
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: Ternary complex of the J-K-St region of EMCV IRES with eIF4A and eIF4G-HEAT1
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weight
IDEntity assembly-IDValue (°)Experimental value
110.1 MDaNO
21NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESC8H18N2O4S1
2150 mMpotassium chlorideKCl1
32 mMmagnesium chlorideMgCl21
40.1 mMATPAdenosine triphosphateC10H16N5O13P31
51 mMDTTC4H10O2S21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Details: The microscope model is the JEOL's "JEM-Z320FHC", the custom-built model equipped with a helium-cooled specimen stage.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 3.4 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 71.1 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 6194
EM imaging opticsEnergyfilter name: In-column Omega Filter / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatchparticle selection
2SerialEMimage acquisition
4GctfCTF correction
7UCSF Chimeramodel fitting
8Cootmodel fitting
10PHENIXmodel refinement
11RELION3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 947392
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 255256 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0147457
ELECTRON MICROSCOPYf_angle_d1.33410527
ELECTRON MICROSCOPYf_dihedral_angle_d17.2471858
ELECTRON MICROSCOPYf_chiral_restr0.0421275
ELECTRON MICROSCOPYf_plane_restr0.005979

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