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- EMDB-1122: Visualizing tmRNA entry into a stalled ribosome. -

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Basic information

Entry
Database: EMDB / ID: EMD-1122
TitleVisualizing tmRNA entry into a stalled ribosome.
Map datathis is a ribosome consisting 70S + Psite tRNA + mRNA + EF-Tu.GDP + alanylated tmRNA + SmpB + Kirromycin + S1
Sample
  • Sample: 70S-Psite tRNA - mRNA - EF-Tu.GDP - alanylated tmRNA - SmpB - Kirromycin - S1
  • Complex: 70S Ribosome
  • RNA: tRNA
  • Protein or peptide: EF-Tu
  • Protein or peptide: SmpB
  • Ligand: tmRNA
Function / homology
Function and homology information


trans-translation / protein-synthesizing GTPase / translation elongation factor activity / GTPase activity / GTP binding / RNA binding / cytosol
Similarity search - Function
SsrA-binding protein / SsrA-binding protein, conserved site / Small protein B / SmpB protein / SsrA-binding 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 / : ...SsrA-binding protein / SsrA-binding protein, conserved site / Small protein B / SmpB protein / SsrA-binding 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 / 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
SsrA-binding protein / Elongation factor Tu-A
Similarity search - Component
Biological speciesThermus thermophilus HB8 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 13.0 Å
AuthorsValle M / Gillet R / Kaur S / Henne A / Ramakrishnan V / Frank J
CitationJournal: Science / Year: 2003
Title: Visualizing tmRNA entry into a stalled ribosome.
Authors: Mikel Valle / Reynald Gillet / Sukhjit Kaur / Anke Henne / V Ramakrishnan / Joachim Frank /
Abstract: Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation ...Bacterial ribosomes stalled on defective messenger RNAs (mRNAs) are rescued by tmRNA, an approximately 300-nucleotide-long molecule that functions as both transfer RNA (tRNA) and mRNA. Translation then switches from the defective message to a short open reading frame on tmRNA that tags the defective nascent peptide chain for degradation. However, the mechanism by which tmRNA can enter and move through the ribosome is unknown. We present a cryo-electron microscopy study at approximately 13 to 15 angstroms of the entry of tmRNA into the ribosome. The structure reveals how tmRNA could move through the ribosome despite its complicated topology and also suggests roles for proteins S1 and SmpB in the function of tmRNA.
History
DepositionMar 24, 2005-
Header (metadata) releaseApr 11, 2005-
Map releaseApr 11, 2005-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 42
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 42
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-1zc8
  • Surface level: 42
  • Imaged by UCSF Chimera
  • Download
  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-1zc8
  • Imaged by Jmol
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1122.gif

Downloads & links

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Map

FileDownload / File: emd_1122.map.gz / Format: CCP4 / Size: 10.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationthis is a ribosome consisting 70S + Psite tRNA + mRNA + EF-Tu.GDP + alanylated tmRNA + SmpB + Kirromycin + S1
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.82 Å/pix.
x 140 pix.
= 394.8 Å		2.82 Å/pix.
x 140 pix.
= 394.8 Å		2.82 Å/pix.
x 140 pix.
= 394.8 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 Level1: 54.899999999999999 / Movie #1: 42
Minimum - Maximum-147.485000000000014 - 313.886000000000024
Average (Standard dev.)2.87458 (±28.770199999999999)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-70-70-70
Dimensions140140140
Spacing140140140
CellA=B=C: 394.8 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.822.822.82
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z394.800394.800394.800
α/β/γ90.00090.00090.000
start NX/NY/NZ-220-220-220
NX/NY/NZ441441441
MAP C/R/S123
start NC/NR/NS-70-70-70
NC/NR/NS140140140
D min/max/mean-147.485313.8862.875

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Supplemental data

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Sample components

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Entire : 70S-Psite tRNA - mRNA - EF-Tu.GDP - alanylated tmRNA - SmpB - Kir...

EntireName: 70S-Psite tRNA - mRNA - EF-Tu.GDP - alanylated tmRNA - SmpB - Kirromycin - S1
Components
  • Sample: 70S-Psite tRNA - mRNA - EF-Tu.GDP - alanylated tmRNA - SmpB - Kirromycin - S1
  • Complex: 70S Ribosome
  • RNA: tRNA
  • Protein or peptide: EF-Tu
  • Protein or peptide: SmpB
  • Ligand: tmRNA

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Supramolecule #1000: 70S-Psite tRNA - mRNA - EF-Tu.GDP - alanylated tmRNA - SmpB - Kir...

SupramoleculeName: 70S-Psite tRNA - mRNA - EF-Tu.GDP - alanylated tmRNA - SmpB - Kirromycin - S1
type: sample / ID: 1000 / Number unique components: 5

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Supramolecule #1: 70S Ribosome

SupramoleculeName: 70S Ribosome / type: complex / ID: 1 / Details: Thermus thermophilus / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)

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Macromolecule #1: tRNA

MacromoleculeName: tRNA / type: rna / ID: 1 / Details: P site / Classification: TRANSFER / Structure: SINGLE STRANDED / Synthetic?: No
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)

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Macromolecule #2: EF-Tu

MacromoleculeName: EF-Tu / type: protein_or_peptide / ID: 2 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)

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Macromolecule #4: SmpB

MacromoleculeName: SmpB / type: protein_or_peptide / ID: 4 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)

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Macromolecule #3: tmRNA

MacromoleculeName: tmRNA / type: ligand / ID: 3 / Recombinant expression: No
Source (natural)Organism: Thermus thermophilus HB8 (bacteria)

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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
Details: 25 mM Hepes-KOH pH 7.5, 30 mM NH4Cl, 7mM MgCl2, 2 mM ATP, 6 mM PEP, 10g/ml PK
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 93 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: two side blotting plunger
Method: Blot for 2 seconds before plunging

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
DateAug 1, 2002
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: ZEISS SCAI / Digitization - Sampling interval: 14 µm / Number real images: 44 / Average electron dose: 15 e/Å2 / Od range: 1.2 / Bits/pixel: 12
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 52000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.7 µm / Nominal defocus min: 1.45 µm / Nominal magnification: 49000
Sample stageSpecimen holder: Cryo transfer / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: defocus groups
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 13.0 Å / Resolution method: OTHER / Software - Name: SPIDER / Number images used: 27644
Final angle assignmentDetails: SPIDER definition

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Atomic model buiding 1

SoftwareName: manual
DetailsProtocol: Rigid Body. The domains were separately fitted by manual docking using program O
RefinementProtocol: RIGID BODY FIT / Target criteria: cross correlation coefficient
Output model

PDB-1zc8:
Coordinates of tmRNA, SmpB, EF-Tu and h44 fitted into Cryo-EM map of the 70S ribosome and tmRNA complex

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