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- EMDB-35041: Cryo-EM structure of the J-K-St region of EMCV IRES in complex wi... -

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Entry
Database: EMDB / ID: EMD-35041
TitleCryo-EM structure of the J-K-St region of EMCV IRES in complex with eIF4G-HEAT1 and eIF4A
Map dataPost-process map
Sample
  • Complex: Ternary complex of the J-K-St region of EMCV IRES with eIF4A and eIF4G-HEAT1
    • Protein or peptide: Eukaryotic initiation factor 4A-I
    • Protein or peptide: Eukaryotic translation initiation factor 4 gamma 1
    • RNA: IRES RNA (J-K-St)
  • Ligand: MAGNESIUM ION
  • Ligand: water
KeywordsTranslation initiation factors / Translation / 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
Eukaryotic initiation factor 4A-I / Eukaryotic translation initiation factor 4 gamma 1
Similarity search - Component
Biological speciesHomo sapiens (human) / Encephalomyocarditis virus
Methodsingle particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsSuzuki H / Fujiyoshi Y / Imai S / Shimada I
Funding support Japan, 2 items
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, 2022-
Header (metadata) releaseAug 2, 2023-
Map releaseAug 2, 2023-
UpdateSep 13, 2023-
Current statusSep 13, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35041.png

Downloads & links

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Map

FileDownload / File: emd_35041.map.gz / Format: CCP4 / Size: 9.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-process map
Voxel sizeX=Y=Z: 1.1475 Å
Density
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.057640046 - 0.11922869
Average (Standard dev.)0.00049246463 (±0.0071004466)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions134134134
Spacing134134134
CellA=B=C: 153.765 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_35041_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35041_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35041_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Ternary complex of the J-K-St region of EMCV IRES with eIF4A and ...

EntireName: Ternary complex of the J-K-St region of EMCV IRES with eIF4A and eIF4G-HEAT1
Components
  • Complex: Ternary complex of the J-K-St region of EMCV IRES with eIF4A and eIF4G-HEAT1
    • Protein or peptide: Eukaryotic initiation factor 4A-I
    • Protein or peptide: Eukaryotic translation initiation factor 4 gamma 1
    • RNA: IRES RNA (J-K-St)
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Ternary complex of the J-K-St region of EMCV IRES with eIF4A and ...

SupramoleculeName: Ternary complex of the J-K-St region of EMCV IRES with eIF4A and eIF4G-HEAT1
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Eukaryotic initiation factor 4A-I

MacromoleculeName: Eukaryotic initiation factor 4A-I / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: RNA helicase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 48.659434 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH HHSSGLEVLF QGPSASQDSR SRDNGPDGME PEGVIESNWN EIVDSFDDMN LSESLLRGIY AYGFEKPSAI QQRAILPCI KGYDVIAQAQ SGTGKTATFA ISILQQIELD LKATQALVLA PTRELAQQIQ KVVMALGDYM GASCHACIGG T NVRAEVQK ...String:
MGSSHHHHHH HHSSGLEVLF QGPSASQDSR SRDNGPDGME PEGVIESNWN EIVDSFDDMN LSESLLRGIY AYGFEKPSAI QQRAILPCI KGYDVIAQAQ SGTGKTATFA ISILQQIELD LKATQALVLA PTRELAQQIQ KVVMALGDYM GASCHACIGG T NVRAEVQK LQMEAPHIIV GTPGRVFDML NRRYLSPKYI KMFVLDEADE MLSRGFKDQI YDIFQKLNSN TQVVLLSATM PS DVLEVTK KFMRDPIRIL VKKEELTLEG IRQFYINVER EEWKLDTLCD LYETLTITQA VIFINTRRKV DWLTEKMHAR DFT VSAMHG DMDQKERDVI MREFRSGSSR VLITTDLLAR GIDVQQVSLV INYDLPTNRE NYIHRIGRGG RFGRKGVAIN MVTE EDKRT LRDIETFYNT SIEEMPLNVA DLI

UniProtKB: Eukaryotic initiation factor 4A-I

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Macromolecule #2: Eukaryotic translation initiation factor 4 gamma 1

MacromoleculeName: Eukaryotic translation initiation factor 4 gamma 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 31.786811 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MNHKVHHHHH HIEGRHMELG TLEDRGEEDA DGSKTQDLFR RVRSILNKLT PQMFQQLMKQ VTQLAIDTEE RLKGVIDLIF EKAISEPNF SVAYANMCRC LMALKVPTTE KPTVTVNFRK LLLNRCQKEF EKDKDDDEVF EKKQKEMDEA ATAEERGRLK E ELEEARDI ...String:
MNHKVHHHHH HIEGRHMELG TLEDRGEEDA DGSKTQDLFR RVRSILNKLT PQMFQQLMKQ VTQLAIDTEE RLKGVIDLIF EKAISEPNF SVAYANMCRC LMALKVPTTE KPTVTVNFRK LLLNRCQKEF EKDKDDDEVF EKKQKEMDEA ATAEERGRLK E ELEEARDI ARRRSLGNIK FIGELFKLKM LTEAIMHDCV VKLLKNHDEE SLECLCRLLT TIGKDLDFEK AKPRMDQYFN QM EKIIKEK KTSSRIRFML QDVLDLRGSN WVP

UniProtKB: Eukaryotic translation initiation factor 4 gamma 1

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Macromolecule #3: IRES RNA (J-K-St)

MacromoleculeName: IRES RNA (J-K-St) / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Encephalomyocarditis virus
Molecular weightTheoretical: 34.838609 KDa
SequenceString:
GGGGCUGAAG GAUGCCCAGA AGGUACCCCA UUGUAUGGGA UCUGAUCUGG GGCCUCGGUG CACAUGCUUU ACAUGUGUUU AGUCGAGGU UAAAAAACGU CUAGGCCCC

GENBANK: GENBANK: NC_001479.1

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 1 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHEPES
150.0 mMKClpotassium chloride
2.0 mMMgCl2magnesium chloride
0.1 mMC10H16N5O13P3ATPAdenosine triphosphate
1.0 mMC4H10O2S2DTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeJEOL CRYO ARM 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 3.4 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
DetailsThe microscope model is the JEOL's "JEM-Z320FHC", the custom-built model equipped with a helium-cooled specimen stage.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 2 / Number real images: 6194 / Average exposure time: 8.0 sec. / Average electron dose: 71.1 e/Å2

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Image processing

Particle selectionNumber selected: 947392
Startup modelType of model: OTHER
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 5 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.76 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 255256
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-8huj:
Cryo-EM structure of the J-K-St region of EMCV IRES in complex with eIF4G-HEAT1 and eIF4A

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