[English] 日本語
Yorodumi- EMDB-1652: Cryo-EM structure of the mammalian Sec61 complex bound to the act... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-1652 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of the mammalian Sec61 complex bound to the actively translating wheat germ 80S ribosome | |||||||||
Map data | The map represents an actively translating wheat germ ribosome with P-site tRNA and a nascent peptide containing the signal anchor sequence of DPAP-B (Dipeptidylaminopeptidase B) bound to the mammalian (Canis familiaris) Sec61 complex. | |||||||||
Sample |
| |||||||||
Keywords | Ribosome / protein exit tunnel / cotranslational protein translocation / protein conducting channel / signal sequence | |||||||||
Function / homology | Function and homology information Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / endoplasmic reticulum Sec complex / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein-transporting ATPase activity / protein insertion into ER membrane ...Insertion of tail-anchored proteins into the endoplasmic reticulum membrane / membrane docking / endoplasmic reticulum Sec complex / pronephric nephron development / cotranslational protein targeting to membrane / Ssh1 translocon complex / Sec61 translocon complex / protein targeting to ER / protein-transporting ATPase activity / protein insertion into ER membrane / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / post-translational protein targeting to membrane, translocation / 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) / L13a-mediated translational silencing of Ceruloplasmin expression / preribosome, large subunit precursor / protein transmembrane transporter activity / regulation of translational fidelity / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / guanyl-nucleotide exchange factor activity / ribosomal large subunit biogenesis / phospholipid binding / ribosome binding / cytosolic large ribosomal subunit / cytoplasmic translation / rRNA binding / structural constituent of ribosome / mRNA binding / endoplasmic reticulum membrane / endoplasmic reticulum / RNA binding / nucleus / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | Canis lupus familiaris (dog) | |||||||||
Method | single particle reconstruction / cryo EM / negative staining / Resolution: 6.48 Å | |||||||||
Authors | Becker 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 | |||||||||
Citation | Journal: 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_1652.map.gz | 17.3 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-1652-v30.xml emd-1652.xml | 12.8 KB 12.8 KB | Display Display | EMDB header |
Images | EMD-1652.jpg | 112.3 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-1652 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-1652 | HTTPS FTP |
-Validation report
Summary document | emd_1652_validation.pdf.gz | 253.6 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_1652_full_validation.pdf.gz | 252.8 KB | Display | |
Data in XML | emd_1652_validation.xml.gz | 7.1 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1652 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-1652 | HTTPS FTP |
-Related structure data
Related structure data | 2wwbMC 1651C 1667C 1668C 1669C 2ww9C 2wwaC M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_1652.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | The map represents an actively translating wheat germ ribosome with P-site tRNA and a nascent peptide containing the signal anchor sequence of DPAP-B (Dipeptidylaminopeptidase B) bound to the mammalian (Canis familiaris) Sec61 complex. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.2375 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : Actively translating wheat germ (O. sativa) 80S ribosome programm...
Entire | Name: Actively translating wheat germ (O. sativa) 80S ribosome programmed with a nascent polypeptide chain containing a P-site tRNA and the type I signal anchor sequence of DPAP-B ...Name: Actively translating wheat germ (O. sativa) 80S ribosome programmed with a nascent polypeptide chain containing a P-site tRNA and the type I signal anchor sequence of DPAP-B (Dipeptidylaminopeptidase B) bound to the mammalian (Canis familiaris) Sec61 complex. |
---|---|
Components |
|
-Supramolecule #1000: Actively translating wheat germ (O. sativa) 80S ribosome programm...
Supramolecule | Name: Actively translating wheat germ (O. sativa) 80S ribosome programmed with a nascent polypeptide chain containing a P-site tRNA and the type I signal anchor sequence of DPAP-B ...Name: Actively translating wheat germ (O. sativa) 80S ribosome programmed with a nascent polypeptide chain containing a P-site tRNA and the type I signal anchor sequence of DPAP-B (Dipeptidylaminopeptidase B) bound to the mammalian (Canis familiaris) Sec61 complex. type: sample / ID: 1000 Details: 80S ribosomes and the digitonin solubilized Sec61 complex were reconstituted in vitro by adding 3 pmol of ribosome and Ssh1 complex in 10 fold molar excess Oligomeric state: 80S Ribosome bound to one copy of the heterotrimeric Sec61 complex Number unique components: 2 |
---|---|
Molecular weight | Experimental: 4.2 MDa / Theoretical: 4.2 MDa / Method: Known for 80S ribosomes |
-Supramolecule #1: Wheat germ (Triticum aestivum) 80 ribosome
Supramolecule | Name: Wheat germ (Triticum aestivum) 80 ribosome / type: complex / ID: 1 / Name.synonym: Wheat germ 80S ribosome / Ribosome-details: ribosome-eukaryote: ALL |
---|---|
Molecular weight | Experimental: 4.2 MDa / Theoretical: 4.2 MDa |
-Macromolecule #1: Mammalian Sec61 complex
Macromolecule | Name: Mammalian Sec61 complex / type: protein_or_peptide / ID: 1 / Name.synonym: Mammalian Sec61 complex / Number of copies: 1 / Oligomeric state: Heterotrimer / Recombinant expression: No |
---|---|
Source (natural) | Organism: Canis lupus familiaris (dog) / synonym: Dog / Tissue: Pancreas / Organelle: Endoplasmic reticulum (ER) membrane / Location in cell: Endoplasmic reticulum |
Molecular weight | Experimental: 70 KDa / Theoretical: 70 KDa |
-Experimental details
-Structure determination
Method | negative staining, cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.02 mg/mL |
---|---|
Buffer | pH: 7.5 Details: 30 mM HEPES/KOH, pH 7.5 180 mM KOAc, 10 mM Mg(OAc)2, 1 mM DTT,3.5 % (w/v) glycerol 0.3 % (w/v) digitonin |
Staining | Type: NEGATIVE / Details: cryo-EM |
Grid | Details: Quantifoil grids (3/3) with 2 nm carbon on top |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot Method: Blot for 10 seconds before plunging, use 2 layers of filter paper |
-Electron microscopy
Microscope | FEI POLARA 300 |
---|---|
Temperature | Average: 84 K |
Alignment procedure | Legacy - Astigmatism: Objective lens astgnatism was corrected at 100000 times magnification |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Digitization - Sampling interval: 4.76 µm / Number real images: 155 / Average electron dose: 25 e/Å2 / Details: Scanned at 5334 dpi / Od range: 1.2 / Bits/pixel: 16 |
Tilt angle min | 0 |
Tilt angle max | 0 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 38000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.26 mm / Nominal defocus max: 4.7 µm / Nominal defocus min: 1.25 µm / Nominal magnification: 39000 |
Sample stage | Specimen holder: FEI Polara cartridge system / Specimen holder model: OTHER |
Experimental equipment | Model: Tecnai Polara / Image courtesy: FEI Company |
-Image processing
Details | Particles were selected using the program SIGNATURE and visually inspected The entire dataset (321000 particles) was sorted for presence of P-site tRNA and presence of ligand. |
---|---|
CTF correction | Details: Defocus group volumes |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 6.48 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER Details: Final map was filtered between 7-8 Angstroms for better visualization of the Sec61 complex Number images used: 221445 |
Final angle assignment | Details: SPIDER |