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- EMDB-4329: Structure of a partial yeast 48S preinitiation complex with eIF5 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-4329
TitleStructure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (Map C2)
Map dataFor optimal visualization of all tRNA and eIF2 gamma, gauss-filter the map by 1.34 and display it at 0.02 contour level.
Sample
  • Complex: Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (model C2)
Biological speciesKluyveromyces lactis (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsLlacer JL / Hussain T / Gordiyenko Y / Ramakrishnan V
CitationJournal: Elife / Year: 2018
Title: Translational initiation factor eIF5 replaces eIF1 on the 40S ribosomal subunit to promote start-codon recognition.
Authors: Jose Luis Llácer / Tanweer Hussain / Adesh K Saini / Jagpreet Singh Nanda / Sukhvir Kaur / Yuliya Gordiyenko / Rakesh Kumar / Alan G Hinnebusch / Jon R Lorsch / V Ramakrishnan /
Abstract: In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNA in a 'P' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) ...In eukaryotic translation initiation, AUG recognition of the mRNA requires accommodation of Met-tRNA in a 'P' state, which is antagonized by the factor eIF1. eIF5 is a GTPase activating protein (GAP) of eIF2 that additionally promotes stringent AUG selection, but the molecular basis of its dual function was unknown. We present a cryo-electron microscopy (cryo-EM) reconstruction of a yeast 48S pre-initiation complex (PIC), at an overall resolution of 3.0 Å, featuring the N-terminal domain (NTD) of eIF5 bound to the 40S subunit at the location vacated by eIF1. eIF5 interacts with and allows a more accommodated orientation of Met-tRNA. Substitutions of eIF5 residues involved in the eIF5-NTD/tRNA interaction influenced initiation at near-cognate UUG codons and the closed/open PIC conformation in vitro, consistent with direct stabilization of the codon:anticodon duplex by the wild-type eIF5-NTD. The present structure reveals the basis for a key role of eIF5 in start-codon selection.
History
Header (metadata) releaseAug 24, 2016-
DepositionMar 12, 2018-
Map releaseDec 5, 2018-
UpdateDec 5, 2018-
Current statusDec 5, 2018Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4329.png

Downloads & links

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Map

FileDownload / File: emd_4329.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFor optimal visualization of all tRNA and eIF2 gamma, gauss-filter the map by 1.34 and display it at 0.02 contour level.
Voxel sizeX=Y=Z: 1.34 Å
Density
Contour LevelBy AUTHOR: 0.12 / Movie #1: 0.12
Minimum - Maximum-0.598529 - 1.1000043
Average (Standard dev.)-0.0000017575309 (±0.03570563)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 402.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.341.341.34
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z402.000402.000402.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.5991.100-0.000

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Supplemental data

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Half map: #1

Fileemd_4329_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_4329_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Structure of a partial yeast 48S preinitiation complex with eIF5 ...

EntireName: Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (model C2)
Components
  • Complex: Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (model C2)

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Supramolecule #1: Structure of a partial yeast 48S preinitiation complex with eIF5 ...

SupramoleculeName: Structure of a partial yeast 48S preinitiation complex with eIF5 N-terminal domain (model C2)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Kluyveromyces lactis (yeast)
Molecular weightTheoretical: 1.8 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

Concentration0.15 mg/mL
BufferpH: 6.5
Component:
ConcentrationName
20.0 mMMES
5.0 mMMagnessium acetate
80.0 mMPotassium acetate
10.0 mMAmmonium acetate
2.0 mMDithiothreitol (DTT)
0.001 mMZinc acetate
0.6 mMATPAdenosine triphosphate
0.25 mMGDPCP
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 400 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated magnification: 104478 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 78000
Sample stageCooling holder cryogen: NITROGEN
TemperatureMin: 90.0 K / Max: 100.0 K
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: INTEGRATING / Number grids imaged: 3 / Number real images: 3600 / Average exposure time: 1.1 sec. / Average electron dose: 40.0 e/Å2
Details: Images were collected in movie-mode at 32 frames per second
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 706713
CTF correctionSoftware - Name: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4v88

Initial angle assignmentType: NOT APPLICABLE / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Number images used: 137103
DetailsFEI Falcon III

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Atomic model buiding 1

RefinementSpace: RECIPROCAL / Protocol: OTHER / Overall B value: 67 / Target criteria: FSC

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