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- EMDB-5030: GTPase activation of elongation factor EF-Tu by the ribosome duri... -

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Entry
Database: EMDB / ID: EMD-5030
TitleGTPase activation of elongation factor EF-Tu by the ribosome during decoding: a cryo-EM structure of the Thermus thermophilus ribosome in which the ternary complex of EF-Tu, tRNA and guanine nucleotide has been trapped on the ribosome with the antibiotic kirromycin.
Map dataThis is an electron density map of a Thermus thermophilus ribosome complexed with mRNA, P- and E-site tRNA as well as EF-Tu.aatRNA.GDP ternary complex.
Sample
  • Sample: Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin.
  • Complex: ribosome
KeywordsThermus thermophilus / 70S / ribosome / EF-Tu / kirromycin / elongation factor / mRNA / Cryo / EM / GTPase / decoding
Function / homology
Function and homology information


translation elongation factor activity / regulation of translation / large ribosomal subunit / transferase activity / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / 5S rRNA binding / ribosomal large subunit assembly ...translation elongation factor activity / regulation of translation / large ribosomal subunit / transferase activity / ribosomal small subunit biogenesis / ribosomal small subunit assembly / small ribosomal subunit / small ribosomal subunit rRNA binding / 5S rRNA binding / ribosomal large subunit assembly / cytosolic small ribosomal subunit / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / ribonucleoprotein complex / GTPase activity / mRNA binding / GTP binding / RNA binding / zinc ion binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
Translation elongation factor EFTu/EF1A, C-terminal / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / : / Elongation factor Tu C-terminal domain / 30S ribosomal protein Thx / 30S ribosomal protein Thx / Ribosomal protein L1, bacterial-type / 30S ribosomal protein ...Translation elongation factor EFTu/EF1A, C-terminal / Translation elongation factor EFTu/EF1A, bacterial/organelle / Elongation factor Tu, domain 2 / Elongation factor Tu (EF-Tu), GTP-binding domain / : / Elongation factor Tu C-terminal domain / 30S ribosomal protein Thx / 30S ribosomal protein Thx / Ribosomal protein L1, bacterial-type / 30S ribosomal protein / Translation elongation factor EF1A/initiation factor IF2gamma, C-terminal / Ribosomal protein L25, long-form / Ribosomal protein L25, beta domain / Ribosomal protein L25, C-terminal / Ribosomal protein TL5, C-terminal domain / Tr-type G domain, conserved site / : / Translational (tr)-type guanine nucleotide-binding (G) domain signature. / Ribosomal protein S14, type Z / Ribosomal protein L1, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L1 / Translation elongation factor EFTu-like, domain 2 / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L11, bacterial-type / Elongation factor Tu domain 2 / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal protein L31 type A / Translational (tr)-type GTP-binding domain / Elongation factor Tu GTP binding domain / Translational (tr)-type guanine nucleotide-binding (G) domain profile. / Ribosomal protein L31 signature. / Ribosomal protein L31 / Ribosomal protein L31 superfamily / Ribosomal protein L31 / Ribosomal protein L11, conserved site / Ribosomal protein L11 signature. / : / Ribosomal protein L9 signature. / Ribosomal protein L9, bacteria/chloroplast / Ribosomal protein L9, C-terminal / Ribosomal protein L9, C-terminal domain / Ribosomal protein L9, C-terminal domain superfamily / Ribosomal protein L11, N-terminal / Ribosomal protein L11, N-terminal domain / 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, RNA binding domain / Ribosomal L25p family / Ribosomal protein L25 / Ribosomal protein L36 signature. / Ribosomal protein L32p, bacterial type / Ribosomal protein L25/Gln-tRNA synthetase, N-terminal / Ribosomal protein L25/Gln-tRNA synthetase, anti-codon-binding domain superfamily / Ribosomal protein L9, N-terminal domain superfamily / Ribosomal protein L9 / Ribosomal protein L9, N-terminal / Ribosomal protein L9, N-terminal domain / Ribosomal protein L33, conserved site / Ribosomal protein L33 signature. / Ribosomal protein L35, conserved site / Ribosomal protein L35 signature. / : / Ribosomal protein L28 / Ribosomal protein L35, non-mitochondrial / Ribosomal protein L5, bacterial-type / Ribosomal protein L18, bacterial-type / : / Ribosomal protein L6, bacterial-type / Ribosomal protein S3, bacterial-type / Ribosomal protein S19, bacterial-type / Ribosomal protein L9/RNase H1, N-terminal / Ribosomal protein S13, bacterial-type / Ribosomal protein S6, conserved site / Ribosomal protein S6 signature. / Ribosomal protein S7, bacterial/organellar-type / Ribosomal protein S9, bacterial/plastid / Ribosomal protein S11, bacterial-type / Ribosomal protein S20 / Ribosomal protein S20 superfamily / Ribosomal protein S20 / Ribosomal protein L36 / Ribosomal protein L36 superfamily / Ribosomal protein L36 / Ribosomal protein S4, bacterial-type / Ribosomal protein S14/S29 / Ribosomal protein L19, conserved site / 30S ribosomal protein S17 / Ribosomal protein L19 signature. / Ribosomal protein S5, bacterial-type / Ribosomal protein L27, conserved site / Ribosomal protein L27 signature. / Ribosomal protein S6, plastid/chloroplast / Ribosomal protein L20 signature. / Ribosomal protein L22, bacterial/chloroplast-type
Similarity search - Domain/homology
Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein uL11 / Elongation factor Tu-B / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL13 ...Small ribosomal subunit protein uS8 / Small ribosomal subunit protein uS14 / Small ribosomal subunit protein uS17 / Large ribosomal subunit protein uL6 / Small ribosomal subunit protein uS7 / Large ribosomal subunit protein bL33 / Large ribosomal subunit protein uL11 / Elongation factor Tu-B / Large ribosomal subunit protein uL2 / Large ribosomal subunit protein uL13 / Large ribosomal subunit protein uL16 / Large ribosomal subunit protein bL19 / Large ribosomal subunit protein bL20 / Large ribosomal subunit protein bL21 / Large ribosomal subunit protein bL27 / Large ribosomal subunit protein bL28 / Small ribosomal subunit protein uS9 / Large ribosomal subunit protein bL32 / Large ribosomal subunit protein bL34 / Large ribosomal subunit protein bL35 / Small ribosomal subunit protein uS2 / Small ribosomal subunit protein uS3 / Small ribosomal subunit protein uS4 / Small ribosomal subunit protein uS11 / Small ribosomal subunit protein uS13 / Small ribosomal subunit protein bS20 / Small ribosomal subunit protein uS12 / Elongation factor Tu-A / Small ribosomal subunit protein uS10 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein uL4 / Large ribosomal subunit protein uL23 / Small ribosomal subunit protein uS19 / Large ribosomal subunit protein uL22 / Large ribosomal subunit protein uL29 / 30S ribosomal protein S17 / Large ribosomal subunit protein uL14 / Large ribosomal subunit protein uL24 / Large ribosomal subunit protein uL5 / 30S ribosomal protein S14 type Z / 30S ribosomal protein S8 / 50S ribosomal protein L6 / Large ribosomal subunit protein uL18 / Small ribosomal subunit protein uS5 / Large ribosomal subunit protein uL30 / Large ribosomal subunit protein uL15 / Large ribosomal subunit protein bL25 / Small ribosomal subunit protein bTHX / Small ribosomal subunit protein uS15 / Large ribosomal subunit protein bL31 / Small ribosomal subunit protein bS16 / Large ribosomal subunit protein uL1 / Small ribosomal subunit protein bS6 / Small ribosomal subunit protein bS18 / Large ribosomal subunit protein bL9 / Large ribosomal subunit protein bL36 / Large ribosomal subunit protein bL17
Similarity search - Component
Biological speciesThermus thermophilus (bacteria)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 6.4 Å
AuthorsSchuette J-C / Murphy F / Kelley AC / Weir J / Giesebrecht J / Connelll SR / Loerke J / Mielke T / Zhang W / Penczek PA ...Schuette J-C / Murphy F / Kelley AC / Weir J / Giesebrecht J / Connelll SR / Loerke J / Mielke T / Zhang W / Penczek PA / Ramakrishnan V / Spahn CMT
CitationJournal: EMBO J / Year: 2009
Title: GTPase activation of elongation factor EF-Tu by the ribosome during decoding.
Authors: Jan-Christian Schuette / Frank V Murphy / Ann C Kelley / John R Weir / Jan Giesebrecht / Sean R Connell / Justus Loerke / Thorsten Mielke / Wei Zhang / Pawel A Penczek / V Ramakrishnan / Christian M T Spahn /
Abstract: We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), ...We have used single-particle reconstruction in cryo-electron microscopy to determine a structure of the Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin. This represents the state in the decoding process just after codon recognition by tRNA and the resulting GTP hydrolysis by EF-Tu, but before the release of EF-Tu from the ribosome. Progress in sample purification and image processing made it possible to reach a resolution of 6.4 A. Secondary structure elements in tRNA, EF-Tu and the ribosome, and even GDP and kirromycin, could all be visualized directly. The structure reveals a complex conformational rearrangement of the tRNA in the A/T state and the interactions with the functionally important switch regions of EF-Tu crucial to GTP hydrolysis. Thus, the structure provides insights into the molecular mechanism of signalling codon recognition from the decoding centre of the 30S subunit to the GTPase centre of EF-Tu.
History
DepositionOct 30, 2008-
Header (metadata) releaseDec 9, 2008-
Map releaseMay 5, 2009-
UpdateJul 23, 2014-
Current statusJul 23, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 3.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 3.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-4v68
  • Surface level: 3.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5030_1.tif

Downloads & links

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Map

FileDownload / File: emd_5030.map.gz / Format: CCP4 / Size: 100.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is an electron density map of a Thermus thermophilus ribosome complexed with mRNA, P- and E-site tRNA as well as EF-Tu.aatRNA.GDP ternary complex.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.26 Å/pix.
x 300 pix.
= 378. Å		1.26 Å/pix.
x 300 pix.
= 378. Å		1.26 Å/pix.
x 300 pix.
= 378. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.26 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 3.5 / Movie #1: 3.2
Minimum - Maximum-4.41906786 - 17.496101379999999
Average (Standard dev.)-0.00041693 (±0.95849848)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-150-150-150
Dimensions300300300
Spacing300300300
CellA=B=C: 378.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.261.261.26
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z378.000378.000378.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-127-127-127
NX/NY/NZ255255255
MAP C/R/S123
start NC/NR/NS-150-150-150
NC/NR/NS300300300
D min/max/mean-4.41917.496-0.000

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Supplemental data

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Sample components

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Entire : Thermus thermophilus ribosome in which the ternary complex of elo...

EntireName: Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin.
Components
  • Sample: Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin.
  • Complex: ribosome

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Supramolecule #1000: Thermus thermophilus ribosome in which the ternary complex of elo...

SupramoleculeName: Thermus thermophilus ribosome in which the ternary complex of elongation factor Tu (EF-Tu), tRNA and guanine nucleotide has been trapped on the ribosome using the antibiotic kirromycin.
type: sample / ID: 1000
Details: The sample was purified using a His-Tag on the ternary complex, which resulted in high occupancy of the ribosome with EF-Tu ternary complex.
Oligomeric state: 70S ribosome with EF-Tu ternary complex, tRNAs and mRNA
Number unique components: 7

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Supramolecule #1: ribosome

SupramoleculeName: ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Thermus thermophilus (bacteria)

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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

StainingType: NEGATIVE
Details: Cryo-EM in vitreous ice. A VITROBOT cryo-plunger was used to prepare grids.
GridDetails: Quantifoil grids.
VitrificationCryogen name: METHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: VITROBOT

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Electron microscopy

MicroscopeFEI TECNAI F30
Detailslow-dose conditions
Image recordingDigitization - Scanner: PRIMESCAN / Number real images: 452 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal magnification: 39000
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN HELIUM
Experimental equipment
Model: Tecnai F30 / Image courtesy: FEI Company

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Image processing

Details586688 individual particle images were obtained from 452 micrographs. Multi-particle refinement resulted in maps with different subconformations. The deposited map represents the most prevalent conformation, based on 323688 particle images.
Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.4 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 323688

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Atomic model buiding 1

Initial modelPDB ID:

1vsa
PDB Unreleased entry

Detailsfitting of molecular models was performed using SITUS and SPIDER.
RefinementSpace: REAL
Output model

PDB-4v68:
T. thermophilus 70S ribosome in complex with mRNA, tRNAs and EF-Tu.GDP.kirromycin ternary complex, fitted to a 6.4 A Cryo-EM map.

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Atomic model buiding 2

Initial modelPDB ID:

2hgu
PDB Unreleased entry

DetailsProtein L11.
RefinementSpace: REAL
Output model

PDB-4v68:
T. thermophilus 70S ribosome in complex with mRNA, tRNAs and EF-Tu.GDP.kirromycin ternary complex, fitted to a 6.4 A Cryo-EM map.

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Atomic model buiding 3

Initial modelPDB ID:

2j00
PDB Unreleased entry

RefinementSpace: REAL
Output model

PDB-4v68:
T. thermophilus 70S ribosome in complex with mRNA, tRNAs and EF-Tu.GDP.kirromycin ternary complex, fitted to a 6.4 A Cryo-EM map.

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Atomic model buiding 4

Initial modelPDB ID:

2j01
PDB Unreleased entry

RefinementSpace: REAL
Output model

PDB-4v68:
T. thermophilus 70S ribosome in complex with mRNA, tRNAs and EF-Tu.GDP.kirromycin ternary complex, fitted to a 6.4 A Cryo-EM map.

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Atomic model buiding 5

Initial modelPDB ID:

1ha3

DetailsWe used the coordinates of the EF-Tu.aatRNA.GDP ternary complex stalled with aurodox.
RefinementSpace: REAL
Output model

PDB-4v68:
T. thermophilus 70S ribosome in complex with mRNA, tRNAs and EF-Tu.GDP.kirromycin ternary complex, fitted to a 6.4 A Cryo-EM map.

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