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- EMDB-4265: Human cap-dependent 48S pre-initiation complex -

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Basic information

Entry
Database: EMDB / ID: EMD-4265
TitleHuman cap-dependent 48S pre-initiation complex
Map data48S pre-initiation complex with eIF2, initiator-tRNA and eIF4B
Sample
  • Complex: human cap-dependent 48S translation pre-initiation complex
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.6 Å
AuthorsSchaffitzel C
CitationJournal: Nucleic Acids Res / Year: 2018
Title: Structure of a human cap-dependent 48S translation pre-initiation complex.
Authors: Boris Eliseev / Lahari Yeramala / Alexander Leitner / Manikandan Karuppasamy / Etienne Raimondeau / Karine Huard / Elena Alkalaeva / Ruedi Aebersold / Christiane Schaffitzel /
Abstract: Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load ...Eukaryotic translation initiation is tightly regulated, requiring a set of conserved initiation factors (eIFs). Translation of a capped mRNA depends on the trimeric eIF4F complex and eIF4B to load the mRNA onto the 43S pre-initiation complex comprising 40S and initiation factors 1, 1A, 2, 3 and 5 as well as initiator-tRNA. Binding of the mRNA is followed by mRNA scanning in the 48S pre-initiation complex, until a start codon is recognised. Here, we use a reconstituted system to prepare human 48S complexes assembled on capped mRNA in the presence of eIF4B and eIF4F. The highly purified h-48S complexes are used for cross-linking/mass spectrometry, revealing the protein interaction network in this complex. We report the electron cryo-microscopy structure of the h-48S complex at 6.3 Å resolution. While the majority of eIF4B and eIF4F appear to be flexible with respect to the ribosome, additional density is detected at the entrance of the 40S mRNA channel which we attribute to the RNA-recognition motif of eIF4B. The eight core subunits of eIF3 are bound at the 40S solvent-exposed side, as well as the subunits eIF3d, eIF3b and eIF3i. elF2 and initiator-tRNA bound to the start codon are present at the 40S intersubunit side. This cryo-EM structure represents a molecular snap-shot revealing the h-48S complex following start codon recognition.
History
DepositionJan 16, 2018-
Header (metadata) releaseMar 7, 2018-
Map releaseMar 7, 2018-
UpdateMar 14, 2018-
Current statusMar 14, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4265.png

Downloads & links

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Map

FileDownload / File: emd_4265.map.gz / Format: CCP4 / Size: 34.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation48S pre-initiation complex with eIF2, initiator-tRNA and eIF4B
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.5 Å/pix.
x 208 pix.
= 520. Å		2.5 Å/pix.
x 208 pix.
= 520. Å		2.5 Å/pix.
x 208 pix.
= 520. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.5 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.047 / Movie #1: 0.06
Minimum - Maximum-0.28490633 - 0.52128345
Average (Standard dev.)-0.000030707484 (±0.023573179)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions208208208
Spacing208208208
CellA=B=C: 520.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.52.52.5
M x/y/z208208208
origin x/y/z0.0000.0000.000
length x/y/z520.000520.000520.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS208208208
D min/max/mean-0.2850.521-0.000

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Supplemental data

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Sample components

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Entire : human cap-dependent 48S translation pre-initiation complex

EntireName: human cap-dependent 48S translation pre-initiation complex
Components
  • Complex: human cap-dependent 48S translation pre-initiation complex

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Supramolecule #1: human cap-dependent 48S translation pre-initiation complex

SupramoleculeName: human cap-dependent 48S translation pre-initiation complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#50
Details: This map comprises human 40S, mRNA, initiator tRNA, eIF2 and eIF4B RRM obtained from multiple sources as described in the publication.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 2.0 MDa

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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
Details: 20 mM Tris HCl, 50 mM KOAc, 2.5 mM MgCl2, 2 mM DTT, 0.25 mM spermidine 0.25 mM GMPPNP
GridModel: Quantifoil R2/2
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 112000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4)
Startup modelType of model: EMDB MAP
EMDB ID:

3019

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 70283
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient

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