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- EMDB-1667: Cryo-EM structure of the active yeast Ssh1 complex bound to the p... -

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Entry
Database: EMDB / ID: EMD-1667
TitleCryo-EM structure of the active yeast Ssh1 complex bound to the programmed yeast 80S ribosome bearing a P-site tRNA
Map dataThis map represents a yeast 80S ribosome bearing a nascent chain of the first 120 amino acid of the type I signal anchor membrane protein DPAP-B, a tRNA in the P-site and the yeast Ssh complex bound at the exit tunnel.
Sample
  • Sample: An active yeast Ssh1 complex bound to a translating yeast ribosome
  • Complex: Yeast 80S ribosome bound to the yeast Ssh1 complex
  • Ligand: Ssh1 complex
KeywordsRibosome / protein exit tunnel / cotranslational protein translocation / protein conducting channel / signal sequence
Function / homology
Function and homology information


translocon complex / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / rough endoplasmic reticulum membrane / Ssh1 translocon complex / Sec61 translocon complex / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / SRP-dependent cotranslational protein targeting to membrane / post-translational protein targeting to membrane, translocation / nuclear inner membrane ...translocon complex / Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / rough endoplasmic reticulum membrane / Ssh1 translocon complex / Sec61 translocon complex / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / SRP-dependent cotranslational protein targeting to membrane / post-translational protein targeting to membrane, translocation / nuclear inner membrane / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / Formation of a pool of free 40S subunits / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / protein transmembrane transporter activity / regulation of translational fidelity / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ribosomal large subunit biogenesis / guanyl-nucleotide exchange factor activity / ribosome binding / ribosomal large subunit assembly / cytosolic large ribosomal subunit / cytoplasmic translation / rRNA binding / structural constituent of ribosome / mRNA binding / structural molecule activity / endoplasmic reticulum membrane / endoplasmic reticulum / RNA binding / membrane / nucleus / cytosol / cytoplasm
Similarity search - Function
Protein transport Sec61-beta/Sbh / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / Protein secE/sec61-gamma signature. / Protein secY signature 1. / SecE/Sec61-gamma subunits of protein translocation complex ...Protein transport Sec61-beta/Sbh / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / Protein secE/sec61-gamma signature. / Protein secY signature 1. / SecE/Sec61-gamma subunits of protein translocation complex / Protein translocase complex, SecE/Sec61-gamma subunit / SecY/SEC61-alpha family / SecY domain superfamily / SecY conserved site / SecY / Ribosomal protein L19, eukaryotic / Ribosomal protein L19/L19e conserved site / Ribosomal protein L19e signature. / Ribosomal protein L23/L25, N-terminal / Ribosomal protein L23, N-terminal domain / Ribosomal protein L39e, conserved site / Ribosomal protein L39e signature. / 60S ribosomal protein L19 / 60S ribosomal protein L35 / 60S ribosomal protein L4, C-terminal domain / 60S ribosomal protein L4 C-terminal domain / Ribosomal_L19e / Ribosomal protein L19/L19e / Ribosomal protein L19/L19e, domain 1 / Ribosomal protein L19/L19e superfamily / Ribosomal protein L19e / Ribosomal protein L31e, conserved site / Ribosomal protein L31e signature. / Ribosomal protein L39e / Ribosomal protein L39e domain superfamily / Ribosomal L39 protein / Ribosomal protein L4/L1e, eukaryotic/archaeal, conserved site / Ribosomal protein L1e signature. / Ribosomal protein L31e / Ribosomal protein L31e domain superfamily / Ribosomal protein L31e / Ribosomal_L31e / Ribosomal protein L4, eukaryotic and archaeal type / Ribosomal protein L22/L17, eukaryotic/archaeal / Ribosomal protein L26/L24, eukaryotic/archaeal / Ribosomal proteins L26 eukaryotic, L24P archaeal / Ribosomal protein L23/L25, conserved site / Ribosomal protein L23 signature. / Ribosomal protein L29, conserved site / Ribosomal protein L29 signature. / Ribosomal protein L22/L17, conserved site / Ribosomal protein L22 signature. / Ribosomal L29 protein / Ribosomal protein L29/L35 / Ribosomal protein L29/L35 superfamily / Ribosomal protein L24 signature. / Ribosomal protein L24/L26, conserved site / Ribosomal protein L23 / Ribosomal protein L25/L23 / Ribosomal protein L26/L24, KOW domain / Ribosomal protein L22/L17 / Ribosomal protein L22/L17 superfamily / Ribosomal protein L22p/L17e / Ribosomal protein L4/L1e / Ribosomal protein L4 domain superfamily / Ribosomal protein L4/L1 family / Translation protein SH3-like domain superfamily / Ribosomal protein L23/L15e core domain superfamily / KOW motif / KOW / Ribosomal protein L2, domain 2 / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / 60S ribosomal protein L19-A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL31A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Protein transport protein SSS1 / Sec sixty-one protein homolog ...Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / 60S ribosomal protein L19-A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL31A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Protein transport protein SSS1 / Sec sixty-one protein homolog / 60S ribosomal protein L35-A / Large ribosomal subunit protein uL4B / Protein transport protein SBH2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 8.6 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-
UpdateJul 15, 2011-
Current statusJul 15, 2011Processing site: PDBe / Status: Released

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Structure visualization

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  • Atomic models: PDB-3izd
  • Surface level: 1.5
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  • Atomic modelsPDB-2ww9
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Movie viewer
Structure viewerEM map:
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Supplemental images

Downloads & links

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Map

FileDownload / File: emd_1667.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis map represents a yeast 80S ribosome bearing a nascent chain of the first 120 amino acid of the type I signal anchor membrane protein DPAP-B, a tRNA in the P-site and the yeast Ssh complex bound at the exit tunnel.
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
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-4.08212 - 9.04266
Average (Standard dev.)0.0505784 (±0.670568)
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-4.0829.0430.051

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Supplemental data

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Sample components

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Entire : An active yeast Ssh1 complex bound to a translating yeast ribosome

EntireName: An active yeast Ssh1 complex bound to a translating yeast ribosome
Components
  • Sample: An active yeast Ssh1 complex bound to a translating yeast ribosome
  • Complex: Yeast 80S ribosome bound to the yeast Ssh1 complex
  • Ligand: Ssh1 complex

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Supramolecule #1000: An active yeast Ssh1 complex bound to a translating yeast ribosome

SupramoleculeName: An active yeast Ssh1 complex bound to a translating yeast ribosome
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
Ribosome-details: ribosome-eukaryote: ALL
Molecular weightExperimental: 4.2 MDa / Theoretical: 4.2 MDa

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Macromolecule #1: Ssh1 complex

MacromoleculeName: Ssh1 complex / type: ligand / ID: 1 / Name.synonym: Ssh1 complex / Details: His FLAG-tagged / Number of copies: 1 / Oligomeric state: Heterotrimer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: RGY1592 / synonym: Baker's yeast / Cell: Rough microsomes / Organelle: Endoplasmic reticulum membrane / Location in cell: Endoplasmic reticulum
Molecular weightTheoretical: 71.5 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)

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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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 38000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / 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
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
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: defocus group volumes
Final angle assignmentDetails: SPIDER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 8.6 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 Ssh1 complex
Number images used: 35800
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.

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