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- EMDB-1233: Structure of eEF3 and the mechanism of transfer RNA release from ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1233
TitleStructure of eEF3 and the mechanism of transfer RNA release from the E-site.
Map dataCryo-EM density map of yeast eEF3 bound to translating yeast 80S ribosome
Sample
  • Sample: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
  • Complex: programmed 80S ribosome
  • Protein or peptide: elongation factor 3
Function / homology
Function and homology information


translational elongation / translational termination / translation elongation factor activity / cytosolic ribosome / : / negative regulation of protein phosphorylation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / negative regulation of protein kinase activity / cytoplasmic stress granule / rRNA binding ...translational elongation / translational termination / translation elongation factor activity / cytosolic ribosome / : / negative regulation of protein phosphorylation / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / negative regulation of protein kinase activity / cytoplasmic stress granule / rRNA binding / ribosome / ATP hydrolysis activity / ATP binding
Similarity search - Function
Elongation Factor 3 / Four helical bundle domain / : / : / Four helical bundle domain / HEAT repeat profile. / HEAT, type 2 / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter ...Elongation Factor 3 / Four helical bundle domain / : / : / Four helical bundle domain / HEAT repeat profile. / HEAT, type 2 / ABC transporter-like, conserved site / ABC transporters family signature. / ABC transporter / ABC transporter-like, ATP-binding domain / ATP-binding cassette, ABC transporter-type domain profile. / Armadillo-like helical / Armadillo-type fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Elongation factor 3A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 9.9 Å
AuthorsBeckmann R / Andersen G
CitationJournal: Nature / Year: 2006
Title: Structure of eEF3 and the mechanism of transfer RNA release from the E-site.
Authors: Christian B F Andersen / Thomas Becker / Michael Blau / Monika Anand / Mario Halic / Bharvi Balar / Thorsten Mielke / Thomas Boesen / Jan Skov Pedersen / Christian M T Spahn / Terri Goss ...Authors: Christian B F Andersen / Thomas Becker / Michael Blau / Monika Anand / Mario Halic / Bharvi Balar / Thorsten Mielke / Thomas Boesen / Jan Skov Pedersen / Christian M T Spahn / Terri Goss Kinzy / Gregers R Andersen / Roland Beckmann /
Abstract: Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of ...Elongation factor eEF3 is an ATPase that, in addition to the two canonical factors eEF1A and eEF2, serves an essential function in the translation cycle of fungi. eEF3 is required for the binding of the aminoacyl-tRNA-eEF1A-GTP ternary complex to the ribosomal A-site and has been suggested to facilitate the clearance of deacyl-tRNA from the E-site. Here we present the crystal structure of Saccharomyces cerevisiae eEF3, showing that it consists of an amino-terminal HEAT repeat domain, followed by a four-helix bundle and two ABC-type ATPase domains, with a chromodomain inserted in ABC2. Moreover, we present the cryo-electron microscopy structure of the ATP-bound form of eEF3 in complex with the post-translocational-state 80S ribosome from yeast. eEF3 uses an entirely new factor binding site near the ribosomal E-site, with the chromodomain likely to stabilize the ribosomal L1 stalk in an open conformation, thus allowing tRNA release.
History
DepositionJun 27, 2006-
Header (metadata) releaseJun 27, 2006-
Map releaseOct 12, 2006-
UpdateSep 2, 2011-
Current statusSep 2, 2011Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0004
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0004
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-2ix8
  • Surface level: 0.0004
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-2ix8
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1233.gif

Downloads & links

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Map

FileDownload / File: emd_1233.map.gz / Format: CCP4 / Size: 78.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM density map of yeast eEF3 bound to translating yeast 80S ribosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2 Å/pix.
x 276 pix.
= 552. Å		2 Å/pix.
x 276 pix.
= 552. Å		2 Å/pix.
x 276 pix.
= 552. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel size
XYZ
EMDB info.222
CCP4 map header222
EM Navigator Movie #11.651.651.65
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.0004 / Movie #1: 0.0004
Minimum - Maximum-0.000957181 - 0.00209009
Average (Standard dev.)0.0000123222 (±0.000166518)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-138-138-138
Dimensions276276276
Spacing276276276
CellA=B=C: 552 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z222
M x/y/z276276276
origin x/y/z0.0000.0000.000
length x/y/z552.000552.000552.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-64-64-64
NX/NY/NZ128128128
MAP C/R/S123
start NC/NR/NS-138-138-138
NC/NR/NS276276276
D min/max/mean-0.0010.0020.000

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Supplemental data

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Sample components

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Entire : 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae

EntireName: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
Components
  • Sample: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae
  • Complex: programmed 80S ribosome
  • Protein or peptide: elongation factor 3

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Supramolecule #1000: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae

SupramoleculeName: 80S-RNC-eEF3-AMP-PNP complex from S. cerevisiae / type: sample / ID: 1000
Details: The emerging signal sequence of the ribosome nascent chain (RNC) was saturated using purified trimeric Sec61 complex in order to prevent biased orientation of particles
Oligomeric state: One ribosome binds to one molecule of eEF3
Number unique components: 2
Molecular weightTheoretical: 4.3 MDa

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Supramolecule #1: programmed 80S ribosome

SupramoleculeName: programmed 80S ribosome / type: complex / ID: 1 / Name.synonym: RNC / Details: programmed 80S ribosome with a P-site tRNA / Ribosome-details: ribosome-eukaryote: ALL
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 MDa

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Macromolecule #1: elongation factor 3

MacromoleculeName: elongation factor 3 / type: protein_or_peptide / ID: 1 / Name.synonym: eEF3 / Details: eEF3 with a C-terminal His-Tag and a factor / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Saccharomyces Cerevisiae / Cell: Saccharomyces Cerevisiae / Organelle: cytosol / Location in cell: cytosol
Molecular weightExperimental: 115 KDa / Theoretical: 115 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast) / Recombinant plasmid: pYES2.1 TOPO

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.168 mg/mL
BufferpH: 7.5
Details: 20 mM HEPES, pH 7.5, 10 mM Mg(OAc)2, 150 mM KOAc 1 mM DTT, 0.05% Nikkol, 125 mM Sucrose,0.01 mg ml-1 Cycloheximide, 0.5 mM AMP-PNP, 0.1 mM Neomycin, 0.3 % DesoxyBigChaps
StainingType: NEGATIVE / Details: Cryo-EM, no staining
GridDetails: Quantifoil grids 200 mesh R2/4
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: Blot for 10 seconds before plunging, use 2 layers of filter paper

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Electron microscopy

MicroscopeFEI TECNAI F30
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38900 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.27 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 39000
Sample stageSpecimen holder: Polara Multispecimen Holder / Specimen holder model: OTHER
TemperatureMin: 95 K / Average: 95 K
Alignment procedureLegacy - Astigmatism: objective lens astigmatism was corrected at 100,000 time
DateJan 13, 2005
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.35 µm / Number real images: 141 / Average electron dose: 20 e/Å2 / Od range: 1.2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 37700

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Atomic model buiding 1

SoftwareName: O and Situs
DetailsProtocol: rigid body. The domains were separately fitted by manual docking using program O
RefinementProtocol: RIGID BODY FIT / Target criteria: cross correlation
Output model

PDB-2ix8:
MODEL FOR EEF3 BOUND TO AN 80S RIBOSOME

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