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- EMDB-5015: A 12.6A cryo-EM map of the 80S.eEF2.AlF4-GDP complex -

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Basic information

Entry
Database: EMDB / ID: EMD-5015
TitleA 12.6A cryo-EM map of the 80S.eEF2.AlF4-GDP complex
Map dataCryo-EM reconstruction of 80S.eEF2 complex captured in transition state
Sample
  • Sample: The eEF2-bound 80S ribosome in the presence of aluminum fluoride (AlF4-) and GDP
  • Complex: 80S ribosomeEukaryotic ribosome
KeywordsAlF4- / GDP / GTPase / Ribosome / Translocation
Function / homology
Function and homology information


translation elongation factor activity / GTPase activity / GTP binding / cytoplasm
Similarity search - Function
Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 ...Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / Translation elongation factor EFTu/EF1A, C-terminal / Elongation factor Tu C-terminal domain / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Tr-type G domain, conserved site / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Translation elongation factor EFTu-like, domain 2 / Elongation factor Tu domain 2 / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Small GTP-binding protein domain / Translation protein, beta-barrel domain superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Elongation factor Tu-B
Similarity search - Component
Biological speciesThermomyces lanuginosus (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 12.6 Å
AuthorsSengupta J / Nilsson J / Gursky R / Kjeldgaard M / Nissen P / Frank J
CitationJournal: J Mol Biol / Year: 2008
Title: Visualization of the eEF2-80S ribosome transition-state complex by cryo-electron microscopy.
Authors: Jayati Sengupta / Jakob Nilsson / Richard Gursky / Morten Kjeldgaard / Poul Nissen / Joachim Frank /
Abstract: In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-A cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum ...In an attempt to understand ribosome-induced GTP hydrolysis on eEF2, we determined a 12.6-A cryo-electron microscopy reconstruction of the eEF2-bound 80S ribosome in the presence of aluminum tetrafluoride and GDP, with aluminum tetrafluoride mimicking the gamma-phosphate during hydrolysis. This is the first visualization of a structure representing a transition-state complex on the ribosome. Tight interactions are observed between the factor's G domain and the large ribosomal subunit, as well as between domain IV and an intersubunit bridge. In contrast, some of the domains of eEF2 implicated in small subunit binding display a large degree of flexibility. Furthermore, we find support for a transition-state model conformation of the switch I region in this complex where the reoriented switch I region interacts with a conserved rRNA region of the 40S subunit formed by loops of the 18S RNA helices 8 and 14. This complex is structurally distinct from the eEF2-bound 80S ribosome complexes previously reported, and analysis of this map sheds light on the GTPase-coupled translocation mechanism.
History
DepositionJun 20, 2008-
Header (metadata) releaseNov 19, 2008-
Map releaseMay 5, 2009-
UpdateMar 6, 2013-
Current statusMar 6, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 65.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 65.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5015_1.png

Downloads & links

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Map

FileDownload / File: emd_5015.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of 80S.eEF2 complex captured in transition state
Voxel sizeX=Y=Z: 2.82 Å
Density
Contour LevelBy AUTHOR: 65.200000000000003 / Movie #1: 65.2
Minimum - Maximum-115.040878300000003 - 227.622665409999996
Average (Standard dev.)4.20576191 (±25.471893309999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions130130130
Spacing130130130
CellA=B=C: 366.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.822.822.82
M x/y/z130130130
origin x/y/z0.0000.0000.000
length x/y/z366.600366.600366.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-127-127-127
NX/NY/NZ255255255
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS130130130
D min/max/mean-115.041227.6234.206

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Supplemental data

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Sample components

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Entire : The eEF2-bound 80S ribosome in the presence of aluminum fluoride ...

EntireName: The eEF2-bound 80S ribosome in the presence of aluminum fluoride (AlF4-) and GDP
Components
  • Sample: The eEF2-bound 80S ribosome in the presence of aluminum fluoride (AlF4-) and GDP
  • Complex: 80S ribosomeEukaryotic ribosome

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Supramolecule #1000: The eEF2-bound 80S ribosome in the presence of aluminum fluoride ...

SupramoleculeName: The eEF2-bound 80S ribosome in the presence of aluminum fluoride (AlF4-) and GDP
type: sample / ID: 1000 / Details: AlF4- mimicking the g-phosphate during hydrolysis / Number unique components: 4

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Supramolecule #1: 80S ribosome

SupramoleculeName: 80S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Thermomyces lanuginosus (fungus)

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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: 7.2 / Details: 20 mM Hepes-NH3, 100 mM KCl, 20 mM MgCl2
GridDetails: Quantifoil grid
VitrificationCryogen name: NITROGEN / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: OTHER / Details: Vitrification instrument: Vitrobot

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Single tilt cyro-holder / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingDigitization - Sampling interval: 2.82 µm / Average electron dose: 10 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Segregation in defocus groups and correction in volumes
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Details: Supervised Classification was used / Number images used: 28242
DetailsThe image processing using SPIDER included a 3D projection alignment procedure with correction of the contrast transfer function and enhancement of the high-resolution Fourier amplitudes based on X-ray solution scattering data.

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Atomic model buiding 1

Initial modelPDB ID:

1n0u

DetailsThe domains were separately fitted by manual docking using program O
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: correlation coefficient
Output model

PDB-3dwu:
Transition-state model conformation of the switch I region fitted into the cryo-EM map of the eEF2.80S.AlF4.GDP complex

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