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- EMDB-1767: AAP-stalled wheat germ 80S ribosome -

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Basic information

Entry
Database: EMDB / ID: EMD-1767
TitleAAP-stalled wheat germ 80S ribosome
Map dataThis is a cryo-EM map of a AAP (arginine attenuator peptide)-stalled wheat germ 80S ribosome.
Sample
  • Sample: AAP-stalled wheat germ 80S ribosome
  • Complex: T. aestivum 80S ribosome
KeywordsAntibiotic / ribosome / translation / stalling / arginine attennuator
Biological speciesTriticum aestivum (bread wheat)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 6.7 Å
AuthorsBhushan S / Meyer H / Starosta A / Becker T / Mielke T / Berninghausen O / Sattler M / Wilson D / Beckmann R
CitationJournal: Mol Cell / Year: 2010
Title: Structural basis for translational stalling by human cytomegalovirus and fungal arginine attenuator peptide.
Authors: Shashi Bhushan / Helge Meyer / Agata L Starosta / Thomas Becker / Thorsten Mielke / Otto Berninghausen / Michael Sattler / Daniel N Wilson / Roland Beckmann /
Abstract: Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the ...Specific regulatory nascent chains establish direct interactions with the ribosomal tunnel, leading to translational stalling. Despite a wealth of biochemical data, structural insight into the mechanism of translational stalling in eukaryotes is still lacking. Here we use cryo-electron microscopy to visualize eukaryotic ribosomes stalled during the translation of two diverse regulatory peptides: the fungal arginine attenuator peptide (AAP) and the human cytomegalovirus (hCMV) gp48 upstream open reading frame 2 (uORF2). The C terminus of the AAP appears to be compacted adjacent to the peptidyl transferase center (PTC). Both nascent chains interact with ribosomal proteins L4 and L17 at tunnel constriction in a distinct fashion. Significant changes at the PTC were observed: the eukaryotic-specific loop of ribosomal protein L10e establishes direct contact with the CCA end of the peptidyl-tRNA (P-tRNA), which may be critical for silencing of the PTC during translational stalling. Our findings provide direct structural insight into two distinct eukaryotic stalling processes.
History
DepositionJul 15, 2010-
Header (metadata) releaseJul 23, 2010-
Map releaseOct 13, 2010-
UpdateOct 17, 2012-
Current statusOct 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-1767.jpg

Downloads & links

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Map

FileDownload / File: emd_1767.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a cryo-EM map of a AAP (arginine attenuator peptide)-stalled wheat germ 80S ribosome.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
1.24 Å/pix.
x 368 pix.
= 455.4 Å		1.24 Å/pix.
x 368 pix.
= 455.4 Å		1.24 Å/pix.
x 368 pix.
= 455.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2375 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.172353 - 0.413648
Average (Standard dev.)0.00218062 (±0.0302681)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-184-184-183
Dimensions368368368
Spacing368368368
CellA=B=C: 455.4 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.23751.23751.2375
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z455.400455.400455.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-184-184-183
NX/NY/NZ368368368
MAP C/R/S213
start NC/NR/NS-184-184-183
NC/NR/NS368368368
D min/max/mean-0.1720.4140.002

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Supplemental data

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Sample components

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Entire : AAP-stalled wheat germ 80S ribosome

EntireName: AAP-stalled wheat germ 80S ribosome
Components
  • Sample: AAP-stalled wheat germ 80S ribosome
  • Complex: T. aestivum 80S ribosome

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Supramolecule #1000: AAP-stalled wheat germ 80S ribosome

SupramoleculeName: AAP-stalled wheat germ 80S ribosome / type: sample / ID: 1000 / Details: Single particle / Oligomeric state: One ribosome / Number unique components: 1
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 MDa

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Supramolecule #1: T. aestivum 80S ribosome

SupramoleculeName: T. aestivum 80S ribosome / type: complex / ID: 1 / Name.synonym: Wheat germ ribosome / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Triticum aestivum (bread wheat) / synonym: Bread wheat
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 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

Concentration0.02 mg/mL
BufferpH: 7.5
Details: 30 mM HEPES/KOH, pH 7.5 180 mM KOAc, 10 mM Mg(OAc)2, 0.01 mg/ml cycloheximide, 5 mM Arginine, 1 mM DTT,3.5 % (w/v) glycerol 0.3 % (w/v) digitonin
StainingType: NEGATIVE / Details: Cryo-EM
GridDetails: Quantifoil Grid with 2 nm carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / 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 POLARA 300
Specialist opticsEnergy filter - Name: FEI
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 4.76 µm / Average electron dose: 25 e/Å2
Details: Scanned at 5334 dpi on a Heidelberg Primescan Drum Scanner
Od range: 1.2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 39000
Sample stageSpecimen holder: FEI Polara Cartridge System / Specimen holder model: OTHER
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe nascent polypeptide chain was saturated with mammlian Sec61 (see Becker et al., Science 2009) to avoid orientational bias on the grid.
CTF correctionDetails: SPIDER TF CTS
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.7 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 165000

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