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- EMDB-5658: Cryo-electron microscopy structure of the mammalian 43S preinitia... -

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Basic information

Entry
Database: EMDB / ID: EMD-5658
TitleCryo-electron microscopy structure of the mammalian 43S preinitiation complex bound to DHX29
Map dataReconstruction of the mammalian 43S preinitiation complex bound to DHX29
Sample
  • Sample: Cryo-electron microscopy structure of the mammalian 43S preinitiation complex bound to DHX29
  • Complex: eukaryotic small ribosomal subunit
  • Protein or peptide: eukaryotic initiation factor 3
  • Protein or peptide: eukaryotic initiation factor 2
  • Protein or peptide: eukaryotic initiation factor 1
  • Protein or peptide: eukaryotic initiation factor 1A
  • Protein or peptide: DHX29
  • RNA: Transfer RNA
Keywords43S / preinitiation complex / eIF3 / eIF2 / Met-tRNAiMet / DHX29 / 40S
Function / homology
Function and homology information


positive regulation of mRNA binding / viral translational termination-reinitiation / eukaryotic translation initiation factor 3 complex, eIF3e / eukaryotic translation initiation factor 3 complex, eIF3m / translation reinitiation / IRES-dependent viral translational initiation / multi-eIF complex / eukaryotic translation initiation factor 3 complex / cytoplasmic translational initiation / eukaryotic 43S preinitiation complex ...positive regulation of mRNA binding / viral translational termination-reinitiation / eukaryotic translation initiation factor 3 complex, eIF3e / eukaryotic translation initiation factor 3 complex, eIF3m / translation reinitiation / IRES-dependent viral translational initiation / multi-eIF complex / eukaryotic translation initiation factor 3 complex / cytoplasmic translational initiation / eukaryotic 43S preinitiation complex / formation of cytoplasmic translation initiation complex / eukaryotic 48S preinitiation complex / metal-dependent deubiquitinase activity / Formation of the ternary complex, and subsequently, the 43S complex / regulation of translational initiation / nuclear-transcribed mRNA catabolic process, nonsense-mediated decay / Ribosomal scanning and start codon recognition / Translation initiation complex formation / protein deubiquitination / Formation of a pool of free 40S subunits / GTP hydrolysis and joining of the 60S ribosomal subunit / negative regulation of proteasomal ubiquitin-dependent protein catabolic process / L13a-mediated translational silencing of Ceruloplasmin expression / translation initiation factor binding / negative regulation of translational initiation / translational initiation / translation initiation factor activity / : / positive regulation of translation / PML body / fibrillar center / metallopeptidase activity / ribosome binding / ubiquitinyl hydrolase 1 / cysteine-type deubiquitinase activity / postsynaptic density / cadherin binding / mRNA binding / synapse / chromatin / structural molecule activity / nucleolus / proteolysis / RNA binding / extracellular exosome / nucleoplasm / membrane / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Eukaryotic translation initiation factor 3 subunit H / eIF3h, C-terminal / C-terminal region of eIF3h / Eukaryotic translation initiation factor 3 subunit F / Translation initiation factor 3 complex subunit L / RNA polymerase I-associated factor PAF67 / Eukaryotic translation initiation factor 3 subunit M / eIF3 subunit M, C-terminal helix domain / eIF3 subunit M, C-terminal helix / Eukaryotic translation initiation factor 3 subunit E, C-terminal ...Eukaryotic translation initiation factor 3 subunit H / eIF3h, C-terminal / C-terminal region of eIF3h / Eukaryotic translation initiation factor 3 subunit F / Translation initiation factor 3 complex subunit L / RNA polymerase I-associated factor PAF67 / Eukaryotic translation initiation factor 3 subunit M / eIF3 subunit M, C-terminal helix domain / eIF3 subunit M, C-terminal helix / Eukaryotic translation initiation factor 3 subunit E, C-terminal / Eukaryotic translation initiation factor 3 subunit E / Eukaryotic translation initiation factor 3 subunit E, N-terminal / eIF3 subunit 6 N terminal domain / eIF3 subunit 6 N terminal domain / Eukaryotic translation initiation factor 3 subunit K / Translation initiation factor 3, subunit 12, N-terminal, eukaryotic / Eukaryotic translation initiation factor 3 subunit M eIF3m/COP9 signalosome complex subunit 7 COPS7 / Eukaryotic translation initiation factor 3 subunit C, N-terminal domain / Eukaryotic translation initiation factor 3 subunit C / Eukaryotic translation initiation factor 3 subunit 8 N-terminus / Eukaryotic translation initiation factor 3 subunit A / CSN8/PSMD8/EIF3K / CSN8/PSMD8/EIF3K family / Rpn11/EIF3F, C-terminal / Maintenance of mitochondrial structure and function / motif in proteasome subunits, Int-6, Nip-1 and TRIP-15 / PCI domain / Proteasome component (PCI) domain / PCI domain profile. / JAB1/Mov34/MPN/PAD-1 ubiquitin protease / JAB/MPN domain / JAB1/MPN/MOV34 metalloenzyme domain / MPN domain / MPN domain profile. / Tetratricopeptide-like helical domain superfamily / Armadillo-type fold / Winged helix DNA-binding domain superfamily / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Eukaryotic translation initiation factor 3 subunit F / Eukaryotic translation initiation factor 3 subunit H / Eukaryotic translation initiation factor 3 subunit E / Eukaryotic translation initiation factor 3 subunit A / Eukaryotic translation initiation factor 3 subunit M / Eukaryotic translation initiation factor 3 subunit C / Eukaryotic translation initiation factor 3 subunit K / Eukaryotic translation initiation factor 3 subunit L
Similarity search - Component
Biological speciesOryctolagus cuniculus (rabbit)
Methodsingle particle reconstruction / cryo EM / Resolution: 11.6 Å
AuthorsHashem Y / des Georges A / Dhote V / Langlois R / Liao HY / Grassucci RA / Hellen CUT / Pestova TV / Frank J
CitationJournal: Cell / Year: 2013
Title: Structure of the mammalian ribosomal 43S preinitiation complex bound to the scanning factor DHX29.
Authors: Yaser Hashem / Amedee des Georges / Vidya Dhote / Robert Langlois / Hstau Y Liao / Robert A Grassucci / Christopher U T Hellen / Tatyana V Pestova / Joachim Frank /
Abstract: Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAi(Met)), eukaryotic initiation factor (eIF) 2, ...Eukaryotic translation initiation begins with assembly of a 43S preinitiation complex. First, methionylated initiator methionine transfer RNA (Met-tRNAi(Met)), eukaryotic initiation factor (eIF) 2, and guanosine triphosphate form a ternary complex (TC). The TC, eIF3, eIF1, and eIF1A cooperatively bind to the 40S subunit, yielding the 43S preinitiation complex, which is ready to attach to messenger RNA (mRNA) and start scanning to the initiation codon. Scanning on structured mRNAs additionally requires DHX29, a DExH-box protein that also binds directly to the 40S subunit. Here, we present a cryo-electron microscopy structure of the mammalian DHX29-bound 43S complex at 11.6 Å resolution. It reveals that eIF2 interacts with the 40S subunit via its α subunit and supports Met-tRNAi(Met) in an unexpected P/I orientation (eP/I). The structural core of eIF3 resides on the back of the 40S subunit, establishing two principal points of contact, whereas DHX29 binds around helix 16. The structure provides insights into eukaryote-specific aspects of translation, including the mechanism of action of DHX29.
History
DepositionMay 1, 2013-
Header (metadata) releaseMay 22, 2013-
Map releaseJun 5, 2013-
UpdateJun 5, 2013-
Current statusJun 5, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j8c
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j8c
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5658_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5658.map.gz / Format: CCP4 / Size: 47.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the mammalian 43S preinitiation complex bound to DHX29
Voxel sizeX=Y=Z: 2.245 Å
Density
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.02738628 - 0.11589623
Average (Standard dev.)0.00035334 (±0.00863221)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions234234234
Spacing234234234
CellA=B=C: 525.32996 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.2452.2452.245
M x/y/z234234234
origin x/y/z0.0000.0000.000
length x/y/z525.330525.330525.330
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS234234234
D min/max/mean-0.0270.1160.000

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Supplemental data

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Sample components

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Entire : Cryo-electron microscopy structure of the mammalian 43S preinitia...

EntireName: Cryo-electron microscopy structure of the mammalian 43S preinitiation complex bound to DHX29
Components
  • Sample: Cryo-electron microscopy structure of the mammalian 43S preinitiation complex bound to DHX29
  • Complex: eukaryotic small ribosomal subunit
  • Protein or peptide: eukaryotic initiation factor 3
  • Protein or peptide: eukaryotic initiation factor 2
  • Protein or peptide: eukaryotic initiation factor 1
  • Protein or peptide: eukaryotic initiation factor 1A
  • Protein or peptide: DHX29
  • RNA: Transfer RNA

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Supramolecule #1000: Cryo-electron microscopy structure of the mammalian 43S preinitia...

SupramoleculeName: Cryo-electron microscopy structure of the mammalian 43S preinitiation complex bound to DHX29
type: sample / ID: 1000
Oligomeric state: One 40S binds one eIF3, one eIF2, one Met-tRNAiMet, and one DHX29
Number unique components: 5

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Supramolecule #1: eukaryotic small ribosomal subunit

SupramoleculeName: eukaryotic small ribosomal subunit / type: complex / ID: 1 / Name.synonym: 40S subunit / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: SSU 40S
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Tissue: blood / Cell: reticulocytes
Molecular weightTheoretical: 1.5 MDa

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Macromolecule #1: eukaryotic initiation factor 3

MacromoleculeName: eukaryotic initiation factor 3 / type: protein_or_peptide / ID: 1 / Name.synonym: eIF3 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Tissue: blood / Cell: reticulocytes
Molecular weightTheoretical: 800 KDa

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Macromolecule #2: eukaryotic initiation factor 2

MacromoleculeName: eukaryotic initiation factor 2 / type: protein_or_peptide / ID: 2 / Name.synonym: eIF2 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit / Tissue: blood / Cell: reticulocytes
Molecular weightTheoretical: 150 KDa

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Macromolecule #3: eukaryotic initiation factor 1

MacromoleculeName: eukaryotic initiation factor 1 / type: protein_or_peptide / ID: 3 / Name.synonym: eIF1 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit
Molecular weightTheoretical: 14 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pT7

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Macromolecule #4: eukaryotic initiation factor 1A

MacromoleculeName: eukaryotic initiation factor 1A / type: protein_or_peptide / ID: 4 / Name.synonym: eIF1A / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit
Molecular weightTheoretical: 19 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pT7

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Macromolecule #5: DHX29

MacromoleculeName: DHX29 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: Yes
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: rabbit
Molecular weightTheoretical: 150 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant plasmid: pT7

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Macromolecule #6: Transfer RNA

MacromoleculeName: Transfer RNA / type: rna / ID: 6 / Name.synonym: tRNA / Details: Met-tRNAiMet / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: Yes
Source (natural)Organism: Oryctolagus cuniculus (rabbit) / synonym: Rabbit
Molecular weightTheoretical: 25 KDa

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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

Concentration0.105 mg/mL
BufferpH: 7.5
Details: 20 mM Tris, 100 mM potassium acetate, 2 mM DTT, 2.5 mM magnesium chloride, 0.25 mM spermidine
GridDetails: 300 mesh copper/molybdenum holey carbon-coated Quantifoil 2/4 grid (Quantifoil Micro Tools GmbH) containing an additional continuous thin layer of carbon
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK II / Method: Blot for seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 110 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51570 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: -4.0 µm / Nominal defocus min: -1.0 µm
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
TemperatureAverage: 110 K
DateNov 1, 2012
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 8000 / Average electron dose: 12 e/Å2 / Bits/pixel: 32

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Image processing

CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 11.6 Å / Resolution method: OTHER / Software - Name: Spider, Relion / Number images used: 29000
DetailsThe particles of this reconstruction were obtained after particle sorting using Relion

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Atomic model buiding 1

Initial modelPDB ID:

2xzm
PDB Unreleased entry

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j8c:
Model of the human eIF3 PCI-MPN octamer docked into the 43S EM map

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Atomic model buiding 2

Initial modelPDB ID:

3v11

SoftwareName: Chimera
DetailsDomains D1-D2 of eIF2-alpha were fitted manually in Chimera in order to accommodate them to their density
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j8c:
Model of the human eIF3 PCI-MPN octamer docked into the 43S EM map

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Atomic model buiding 3

Initial modelPDB ID:

3zwl

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3j8c:
Model of the human eIF3 PCI-MPN octamer docked into the 43S EM map

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