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- EMDB-1564: Recognition of Phe-tRNAPhe, Trp-tRNATrp and : a common molecular ... -

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Basic information

Entry
Database: EMDB / ID: EMD-1564
TitleRecognition of Phe-tRNAPhe, Trp-tRNATrp and : a common molecular mechanism for aminoacyl-tRNA selection revealed by cryo-EM
Map dataLeu-tRNA-EFTu-kir-GDP-70S ribosome
Sample
  • Sample: Leu-tRNA-EFTu-GDP-kir-70S ribosome
  • Complex: 70S ribosomeRibosome
Keywordsprotein translation / decoding / elongation factor Tu / ribosome
Function / homology
Function and homology information


guanyl-nucleotide exchange factor complex / guanosine tetraphosphate binding / translational elongation / stringent response / misfolded RNA binding / Group I intron splicing / RNA folding / translational termination / translation elongation factor activity / positive regulation of RNA splicing ...guanyl-nucleotide exchange factor complex / guanosine tetraphosphate binding / translational elongation / stringent response / misfolded RNA binding / Group I intron splicing / RNA folding / translational termination / translation elongation factor activity / positive regulation of RNA splicing / maintenance of translational fidelity / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / rRNA binding / ribosome / structural constituent of ribosome / translation / response to antibiotic / GTPase activity / GTP binding / RNA binding / plasma membrane / cytosol / 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 / Ribosomal protein L11, bacterial-type / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / Ribosomal protein L11, N-terminal / Ribosomal protein L11/L12 / Ribosomal protein L11, C-terminal / Ribosomal protein L11, C-terminal domain superfamily / Ribosomal protein L11/L12, N-terminal domain superfamily / Ribosomal protein L11/L12 / Ribosomal protein L11, N-terminal domain / Ribosomal protein L11, RNA binding domain / Ribosomal protein S12, bacterial-type / Ribosomal protein S12/S23 / Ribosomal protein S12/S23 / Small GTP-binding protein domain / Ribosomal protein S12 signature. / Translation protein, beta-barrel domain superfamily / Nucleic acid-binding, OB-fold / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Elongation factor Tu 1 / Large ribosomal subunit protein uL11 / Small ribosomal subunit protein uS12 / Elongation factor Tu 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 12.0 Å
AuthorsLi W / Agirrezabala X / Lei J / Bouakaz L / Brunelle JL / Ortiz-Meoz RF / Green R / Sanyal S / Ehrenberg M / Frank J
CitationJournal: EMBO J / Year: 2008
Title: Recognition of aminoacyl-tRNA: a common molecular mechanism revealed by cryo-EM.
Authors: Wen Li / Xabier Agirrezabala / Jianlin Lei / Lamine Bouakaz / Julie L Brunelle / Rodrigo F Ortiz-Meoz / Rachel Green / Suparna Sanyal / Måns Ehrenberg / Joachim Frank /
Abstract: The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes ...The accuracy of ribosomal translation is achieved by an initial selection and a proofreading step, mediated by EF-Tu, which forms a ternary complex with aminoacyl(aa)-tRNA. To study the binding modes of different aa-tRNAs, we compared cryo-EM maps of the kirromycin-stalled ribosome bound with ternary complexes containing Phe-tRNA(Phe), Trp-tRNA(Trp), or Leu-tRNA(LeuI). The three maps suggest a common binding manner of cognate aa-tRNAs in their specific binding with both the ribosome and EF-Tu. All three aa-tRNAs have the same 'loaded spring' conformation with a kink and twist between the D-stem and anticodon stem. The three complexes are similarly integrated in an interaction network, extending from the anticodon loop through h44 and protein S12 to the EF-Tu-binding CCA end of aa-tRNA, proposed to signal cognate codon-anticodon interaction to the GTPase centre and tune the accuracy of aa-tRNA selection.
History
DepositionSep 29, 2008-
Header (metadata) releaseSep 29, 2008-
Map releaseJun 1, 2010-
UpdateSep 10, 2014-
Current statusSep 10, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0003
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.0003
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3eq4
  • Surface level: 0.0003
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3eq4
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1564.png

Downloads & links

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Map

FileDownload / File: emd_1564.map.gz / Format: CCP4 / Size: 8.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationLeu-tRNA-EFTu-kir-GDP-70S ribosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.82 Å/pix.
x 130 pix.
= 366.6 Å		2.82 Å/pix.
x 130 pix.
= 366.6 Å		2.82 Å/pix.
x 130 pix.
= 366.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.00045 / Movie #1: 0.0003
Minimum - Maximum-0.000495627 - 0.00147413
Average (Standard dev.)0.0000353844 (±0.000183878)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-65-65-65
Dimensions130130130
Spacing130130130
CellA=B=C: 366.6 Å
α=β=γ: 90 °

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-81-81-81
NX/NY/NZ160160160
MAP C/R/S123
start NC/NR/NS-65-65-65
NC/NR/NS130130130
D min/max/mean-0.0000.0010.000

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Supplemental data

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Sample components

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Entire : Leu-tRNA-EFTu-GDP-kir-70S ribosome

EntireName: Leu-tRNA-EFTu-GDP-kir-70S ribosome
Components
  • Sample: Leu-tRNA-EFTu-GDP-kir-70S ribosome
  • Complex: 70S ribosomeRibosome

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Supramolecule #1000: Leu-tRNA-EFTu-GDP-kir-70S ribosome

SupramoleculeName: Leu-tRNA-EFTu-GDP-kir-70S ribosome / type: sample / ID: 1000 / Details: sedimentation / Oligomeric state: monomeric / Number unique components: 5
Molecular weightExperimental: 2.8 MDa / Theoretical: 2.8 MDa

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Name.synonym: 70S ribosome / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 2.8 MDa / Theoretical: 2.8 MDa

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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

Concentration0.03 mg/mL
BufferpH: 7.3
Details: Polymix buffer( 5 mM potassium phosphate (KH2PO4) pH 7.3, 5 mM NH4Cl, 95 mM KCl, 0.5 mM CaCl2, 8 mM putrescine, 1 mM spermidine, 1 mM DTE, 5 mM magnesium acetate)
StainingType: NEGATIVE / Details: cryo
GridDetails: 200 mesh
VitrificationCryogen name: NITROGEN / Chamber humidity: 90 % / Chamber temperature: 80 K / Instrument: OTHER / Details: Vitrification instrument: vitrobot / Method: Blot for 6 seconds

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49645 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.26 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Specialist opticsEnergy filter - Name: no energy filter
Sample stageSpecimen holder: cartridge / Specimen holder model: OTHER
TemperatureMin: 80.7 K / Max: 80.7 K / Average: 80.7 K
Alignment procedureLegacy - Astigmatism: corrected objective at 100,000 times magnification
Legacy - Electron beam tilt params: no tilt
DateJun 1, 2006
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 7 µm / Number real images: 112 / Average electron dose: 15 e/Å2 / Bits/pixel: 2
Tilt angle min0
Tilt angle max0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: defocus groups, Wiener filter
Final angle assignmentDetails: spider
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 12.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 80000

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Atomic model buiding 1

Initial model(PDB ID:

2avy
PDB Unreleased entry

,

2aw4
PDB Unreleased entry

,

1gix
PDB Unreleased entry

,

1ibm

,

1qza

)
SoftwareName: TNT
DetailsProtocol: real space refinement. 1lBM(1989-1996 fragment)
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-3eq4:
Model of tRNA(Leu)-EF-Tu in the ribosomal pre-accommodated state revealed by cryo-EM

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