+Open data
-Basic information
Entry | Database: PDB / ID: 3j46 | |||||||||
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Title | Structure of the SecY protein translocation channel in action | |||||||||
Components |
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Keywords | RIBOSOME/PROTEIN TRANSPORT / 70S / preprotein translocase / SECYEG / protein translocation channel / nascent chain / RIBOSOME-PROTEIN TRANSPORT complex | |||||||||
Function / homology | Function and homology information protein insertion into membrane from inner side / cell envelope Sec protein transport complex / protein transport by the Sec complex / intracellular protein transmembrane transport / protein-transporting ATPase activity / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / protein transmembrane transporter activity / protein secretion / negative regulation of translational initiation ...protein insertion into membrane from inner side / cell envelope Sec protein transport complex / protein transport by the Sec complex / intracellular protein transmembrane transport / protein-transporting ATPase activity / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / protein transmembrane transporter activity / protein secretion / negative regulation of translational initiation / ribosomal large subunit assembly / intracellular protein transport / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / tRNA binding / rRNA binding / structural constituent of ribosome / translation / membrane / plasma membrane / cytoplasm / cytosol Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 10.1 Å | |||||||||
Authors | Akey, C.W. / Park, E. / Menetret, J.F. / Gumbart, J.C. / Ludtke, S.J. / Li, W. / Whynot, A. / Rapoport, T.A. | |||||||||
Citation | Journal: Nature / Year: 2014 Title: Structure of the SecY channel during initiation of protein translocation. Authors: Eunyong Park / Jean-François Ménétret / James C Gumbart / Steven J Ludtke / Weikai Li / Andrew Whynot / Tom A Rapoport / Christopher W Akey / Abstract: Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during ...Many secretory proteins are targeted by signal sequences to a protein-conducting channel, formed by prokaryotic SecY or eukaryotic Sec61 complexes, and are translocated across the membrane during their synthesis. Crystal structures of the inactive channel show that the SecY subunit of the heterotrimeric complex consists of two halves that form an hourglass-shaped pore with a constriction in the middle of the membrane and a lateral gate that faces the lipid phase. The closed channel has an empty cytoplasmic funnel and an extracellular funnel that is filled with a small helical domain, called the plug. During initiation of translocation, a ribosome-nascent chain complex binds to the SecY (or Sec61) complex, resulting in insertion of the nascent chain. However, the mechanism of channel opening during translocation is unclear. Here we have addressed this question by determining structures of inactive and active ribosome-channel complexes with cryo-electron microscopy. Non-translating ribosome-SecY channel complexes derived from Methanocaldococcus jannaschii or Escherichia coli show the channel in its closed state, and indicate that ribosome binding per se causes only minor changes. The structure of an active E. coli ribosome-channel complex demonstrates that the nascent chain opens the channel, causing mostly rigid body movements of the amino- and carboxy-terminal halves of SecY. In this early translocation intermediate, the polypeptide inserts as a loop into the SecY channel with the hydrophobic signal sequence intercalated into the open lateral gate. The nascent chain also forms a loop on the cytoplasmic surface of SecY rather than entering the channel directly. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 3j46.cif.gz | 430 KB | Display | PDBx/mmCIF format |
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PDB format | pdb3j46.ent.gz | 312.1 KB | Display | PDB format |
PDBx/mmJSON format | 3j46.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 3j46_validation.pdf.gz | 809.4 KB | Display | wwPDB validaton report |
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Full document | 3j46_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | 3j46_validation.xml.gz | 70.9 KB | Display | |
Data in CIF | 3j46_validation.cif.gz | 104.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/j4/3j46 ftp://data.pdbj.org/pub/pdb/validation_reports/j4/3j46 | HTTPS FTP |
-Related structure data
Related structure data | 5693MC 5691C 5692C 3j45C 4v4nC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
-Protein , 4 types, 4 molecules yEGn
#1: Protein | Mass: 47659.371 Da / Num. of mol.: 1 / Mutation: S68C Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: pBAD(MazF)-NC100 / Production host: Escherichia coli (E. coli) / Strain (production host): EP72 / References: UniProt: P0AGA2 |
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#2: Protein | Mass: 6123.306 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: pBAD(MazF)-NC100 / Production host: Escherichia coli (E. coli) / Strain (production host): EP72 / References: UniProt: P0AG96 |
#3: Protein | Mass: 6566.800 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: pBAD(MazF)-NC100 / Production host: Escherichia coli (E. coli) / Strain (production host): EP72 / References: UniProt: P0AG99 |
#4: Protein | Mass: 10780.095 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: pBAD(MazF)-NC100 / Production host: Escherichia coli (E. coli) / Strain (production host): EP72 |
-RNA chain , 2 types, 2 molecules pa
#5: RNA chain | Mass: 24478.502 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) |
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#6: RNA chain | Mass: 24599.748 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) |
-50S ribosomal protein ... , 4 types, 4 molecules 5TUY
#7: Protein | Mass: 24765.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7L0 |
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#8: Protein | Mass: 11222.160 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0ADZ0 |
#9: Protein | Mass: 11208.054 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P60624 |
#10: Protein | Mass: 7286.464 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) / References: UniProt: P0A7M6 |
-23S ribosomal ... , 4 types, 4 molecules 1234
#11: RNA chain | Mass: 20350.104 Da / Num. of mol.: 1 / Fragment: helix 6 - helix 7 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) |
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#12: RNA chain | Mass: 11661.961 Da / Num. of mol.: 1 / Fragment: helix 50 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) |
#13: RNA chain | Mass: 14261.592 Da / Num. of mol.: 1 / Fragment: helix 59 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) |
#14: RNA chain | Mass: 35116.715 Da / Num. of mol.: 1 / Fragment: helix 76 - helix 78 / Source method: isolated from a natural source / Source: (natural) Escherichia coli (E. coli) |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY / Number of used crystals: 1 |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight | Value: 2.5 MDa / Experimental value: NO | ||||||||||||||||||||
Buffer solution | Name: 50 mM Tris-acetate, 10 mM Mg(OAc)2, 80 mM KOAc, 0.06% DDM pH: 7.2 Details: 50 mM Tris-acetate, 10 mM Mg(OAc)2, 80 mM KOAc, 0.06% DDM | ||||||||||||||||||||
Specimen | Conc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Details: 400 mesh Quantifoil holey grids with 2/1 or 1.2/1.2 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Temp: 77 K / Humidity: 95 % Details: Blot 1-2 seconds before plunging into liquid ethane (FEI VITROBOT MARK III). |
-Electron microscopy imaging
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
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Microscopy | Model: FEI TECNAI F20 / Date: Feb 10, 2012 Details: Low dose imaging: automated single particle data collection program from TVIPS was used. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 160 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 42000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2 mm |
Specimen holder | Specimen holder model: OTHER / Specimen holder type: CT3500 / Temperature: 94 K |
Image recording | Electron dose: 20 e/Å2 / Film or detector model: KODAK SO-163 FILM / Details: Tietz 4096 x 4096 CCD |
Image scans | Num. digital images: 4900 |
Radiation wavelength | Relative weight: 1 |
-Processing
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EM software |
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CTF correction | Details: per micrograph | ||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||
3D reconstruction | Method: projection matching / Resolution: 10.1 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 53000 / Nominal pixel size: 2.12 Å / Actual pixel size: 2.12 Å Details: The structure was solved twice: first with a model starting from a 25-Angstrom filtered E. coli ribosome map generated in house, and then a second time using a filtered ribosome model (EMD- ...Details: The structure was solved twice: first with a model starting from a 25-Angstrom filtered E. coli ribosome map generated in house, and then a second time using a filtered ribosome model (EMD-5036). In each case, after convergence, maps from two EMAN2 refinements with different parameters were averaged after alignment in Chimera. Four maps in total were averaged to reduce the noise. Resolution method was FSC at 0.5 cut-off for a comparison between the full experimental 3D density map and a calculated map of the docked E. coli ribosome model (this map was calculated to 7 Angstrom resolution with EMAN). Num. of class averages: 5300 / Symmetry type: POINT | ||||||||||||||||||||
Atomic model building |
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Atomic model building |
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Refinement step | Cycle: LAST
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