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- EMDB-20326: Cryo-EM structure of yeast elongation-competent 80S complex (80S EC) -

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Basic information

Entry
Database: EMDB / ID: EMD-20326
TitleCryo-EM structure of yeast elongation-competent 80S complex (80S EC)
Map dataprimary map
Sample
  • Complex: A cryo-EM map of a yeast elongation-competent 80S complex (80S-EC)
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.1 Å
AuthorsWang J / Chen DH / Puglisi JD
Funding support United States, Sweden, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical SciencesGM011378 United States
Knut and Alice Wallenberg Foundation Sweden
CitationJournal: Nature / Year: 2019
Title: eIF5B gates the transition from translation initiation to elongation.
Authors: Jinfan Wang / Alex G Johnson / Christopher P Lapointe / Junhong Choi / Arjun Prabhakar / Dong-Hua Chen / Alexey N Petrov / Joseph D Puglisi /
Abstract: Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous ...Translation initiation determines both the quantity and identity of the protein that is encoded in an mRNA by establishing the reading frame for protein synthesis. In eukaryotic cells, numerous translation initiation factors prepare ribosomes for polypeptide synthesis; however, the underlying dynamics of this process remain unclear. A central question is how eukaryotic ribosomes transition from translation initiation to elongation. Here we use in vitro single-molecule fluorescence microscopy approaches in a purified yeast Saccharomyces cerevisiae translation system to monitor directly, in real time, the pathways of late translation initiation and the transition to elongation. This transition was slower in our eukaryotic system than that reported for Escherichia coli. The slow entry to elongation was defined by a long residence time of eukaryotic initiation factor 5B (eIF5B) on the 80S ribosome after the joining of individual ribosomal subunits-a process that is catalysed by this universally conserved initiation factor. Inhibition of the GTPase activity of eIF5B after the joining of ribosomal subunits prevented the dissociation of eIF5B from the 80S complex, thereby preventing elongation. Our findings illustrate how the dissociation of eIF5B serves as a kinetic checkpoint for the transition from initiation to elongation, and how its release may be governed by a change in the conformation of the ribosome complex that triggers GTP hydrolysis.
History
DepositionJun 20, 2019-
Header (metadata) releaseJul 24, 2019-
Map releaseJul 24, 2019-
UpdateNov 6, 2019-
Current statusNov 6, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.048
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20326.png

Downloads & links

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Map

FileDownload / File: emd_20326.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationprimary map
Voxel sizeX=Y=Z: 2.53 Å
Density
Contour LevelBy AUTHOR: 0.048 / Movie #1: 0.048
Minimum - Maximum-0.082155645 - 0.16330838
Average (Standard dev.)0.0008557438 (±0.014776892)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 485.76 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.532.532.53
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z485.760485.760485.760
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0820.1630.001

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Supplemental data

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Sample components

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Entire : A cryo-EM map of a yeast elongation-competent 80S complex (80S-EC)

EntireName: A cryo-EM map of a yeast elongation-competent 80S complex (80S-EC)
Components
  • Complex: A cryo-EM map of a yeast elongation-competent 80S complex (80S-EC)

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Supramolecule #1: A cryo-EM map of a yeast elongation-competent 80S complex (80S-EC)

SupramoleculeName: A cryo-EM map of a yeast elongation-competent 80S complex (80S-EC)
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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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:
ConcentrationFormula
30.0 mMHEPES-KOH
3.0 mMMg-acetate
100.0 mMK-acetate
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 294 K / Instrument: LEICA EM GP / Details: Blot 2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 19762 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal magnification: 14500
Sample stageSpecimen holder model: GATAN 626 SINGLE TILT LIQUID NITROGEN CRYO TRANSFER HOLDER
Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 10.0 sec. / Average electron dose: 15.6 e/Å2
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 10.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 7542

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