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- EMDB-1668: Cryo-EM structure of the active yeast 80S ribosome bearing a P-si... -

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Basic information

Entry
Database: EMDB / ID: EMD-1668
TitleCryo-EM structure of the active yeast 80S ribosome bearing a P-site tRNA and with the rRNA expansion segment ES27 in the exit conformation
Map dataThis map represents a yeast 80S ribosome programmed with the first 120 amino acids of the type I signal anchor protein DPAP-B attached to a P-site tRNA. The expansion segment ES27 is in the exit position.
Sample
  • Sample: A programmed yeast ribosome with ES27 in the exit conformation
  • Complex: Yeast 80S ribosome bound to the yeast Ssh1 complex
KeywordsRibosome / protein exit tunnel / cotranslational protein translocation / protein conducting channel / signal sequence
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 7.9 Å
AuthorsBecker T / Mandon E / Bhushan S / Jarasch A / Armache JP / Funes S / Jossinet F / Gumbart J / Mielke T / Berninghausen O ...Becker T / Mandon E / Bhushan S / Jarasch A / Armache JP / Funes S / Jossinet F / Gumbart J / Mielke T / Berninghausen O / Schulten K / Westhof E / Gilmore R / Beckmann R
CitationJournal: Science / Year: 2009
Title: Structure of monomeric yeast and mammalian Sec61 complexes interacting with the translating ribosome.
Authors: Thomas Becker / Shashi Bhushan / Alexander Jarasch / Jean-Paul Armache / Soledad Funes / Fabrice Jossinet / James Gumbart / Thorsten Mielke / Otto Berninghausen / Klaus Schulten / Eric ...Authors: Thomas Becker / Shashi Bhushan / Alexander Jarasch / Jean-Paul Armache / Soledad Funes / Fabrice Jossinet / James Gumbart / Thorsten Mielke / Otto Berninghausen / Klaus Schulten / Eric Westhof / Reid Gilmore / Elisabet C Mandon / Roland Beckmann /
Abstract: The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may ...The trimeric Sec61/SecY complex is a protein-conducting channel (PCC) for secretory and membrane proteins. Although Sec complexes can form oligomers, it has been suggested that a single copy may serve as an active PCC. We determined subnanometer-resolution cryo-electron microscopy structures of eukaryotic ribosome-Sec61 complexes. In combination with biochemical data, we found that in both idle and active states, the Sec complex is not oligomeric and interacts mainly via two cytoplasmic loops with the universal ribosomal adaptor site. In the active state, the ribosomal tunnel and a central pore of the monomeric PCC were occupied by the nascent chain, contacting loop 6 of the Sec complex. This provides a structural basis for the activity of a solitary Sec complex in cotranslational protein translocation.
History
DepositionDec 3, 2009-
Header (metadata) releaseDec 16, 2009-
Map releaseDec 22, 2010-
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: 1.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.2
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1668.gif

1668_EMD_166...

Downloads & links

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Map

FileDownload / File: emd_1668.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis map represents a yeast 80S ribosome programmed with the first 120 amino acids of the type I signal anchor protein DPAP-B attached to a P-site tRNA. The expansion segment ES27 is in the exit position.
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: 1.2 / Movie #1: 1.2
Minimum - Maximum-3.7418 - 8.980309999999999
Average (Standard dev.)0.0514786 (±0.710387)
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-3.7428.9800.051

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Supplemental data

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Sample components

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Entire : A programmed yeast ribosome with ES27 in the exit conformation

EntireName: A programmed yeast ribosome with ES27 in the exit conformation
Components
  • Sample: A programmed yeast ribosome with ES27 in the exit conformation
  • Complex: Yeast 80S ribosome bound to the yeast Ssh1 complex

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Supramolecule #1000: A programmed yeast ribosome with ES27 in the exit conformation

SupramoleculeName: A programmed yeast ribosome with ES27 in the exit conformation
type: sample / ID: 1000
Details: 80S ribosomes and the detergent solubilized Ssh1 complex were reconstituted in vitro by adding 1 pmol of ribosome and Ssh1 complex in 5 fold molar excess
Oligomeric state: 80S Ribosome bound to one copy of the heterotrimeric Ssh1 complex
Number unique components: 2
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 MDa / Method: Known for 80S ribosomes

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Supramolecule #1: Yeast 80S ribosome bound to the yeast Ssh1 complex

SupramoleculeName: Yeast 80S ribosome bound to the yeast Ssh1 complex / type: complex / ID: 1
Name.synonym: Yeast 80S ribosome bound to the yeast Ssh1 complex
Recombinant expression: No / Ribosome-details: ribosome-eukaryote: ALL
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast
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

BufferpH: 7.5
Details: 20 mM HEPES/KOH, pH 7.5 100 mM KOAc, 10 mM Mg(OAc)2, 1.5 mM DTT, 0.1 % (w/v) digitonin
StainingType: NEGATIVE / Details: Cryo-EM
GridDetails: Quantifoil grids (3/3) with 2 nm carbon on top
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: Blot for 10 seconds before plunging, use 2 layer of filter paper

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Electron microscopy

MicroscopeFEI POLARA 300
TemperatureAverage: 84 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 100000 times magnification
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.76 µm / Number real images: 185 / Average electron dose: 25 e/Å2 / Details: Scanned at 5334 dpi / Od range: 1.2 / Bits/pixel: 16
Tilt angle min0
Tilt angle max0
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: 1.2 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

DetailsParticles were selected using the program SIGNATURE and visually inspected. This map resulted from sorting against the ES27 exit position and subsequent sorting for tRNA and the Ssh1 complex and represents the datasubset with ES27 in the exit conformation
CTF correctionDetails: Defocus group volumes
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 7.9 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER
Details: Map was filtered between 8.3 and 10.3 Angstrom to better visualize the rRNA segment ES27 in the exit conformation
Number images used: 69000
Final angle assignmentDetails: SPIDER

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