+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5693 | |||||||||
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Title | Structure of the SecY protein translocation channel in action | |||||||||
Map data | ribosome-nascent chain-SecYEG complex | |||||||||
Sample |
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Keywords | ribosome-channel complex / active SecYEG channel / nascent chain / E. coli 70S ribosome | |||||||||
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 / intracellular protein transport / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / tRNA binding / rRNA binding / structural constituent of ribosome / translation / membrane / plasma membrane / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 10.1 Å | |||||||||
Authors | Park E / Menetret JF / Gumbart JC / Ludtke SJ / Li W / Whynot A / Rapoport TA / Akey CW | |||||||||
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 | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_5693.map.gz | 3 MB | EMDB map data format | |
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Header (meta data) | emd-5693-v30.xml emd-5693.xml | 35.5 KB 35.5 KB | Display Display | EMDB header |
Images | emd_5693_1.jpg | 182.5 KB | ||
Masks | emd_5693_msk_1.map emd_5693_msk_2.map emd_5693_msk_3.map emd_5693_msk_4.map emd_5693_msk_5.map emd_5693_msk_6.map emd_5693_msk_7.map emd_5693_msk_8.map emd_5693_msk_9.map | 27 MB 601 KB 27 MB 263.3 KB 27 MB 27 MB 27 MB 27 MB 4.3 MB | Mask map | |
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5693 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5693 | HTTPS FTP |
-Validation report
Summary document | emd_5693_validation.pdf.gz | 620 KB | Display | EMDB validaton report |
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Full document | emd_5693_full_validation.pdf.gz | 619.5 KB | Display | |
Data in XML | emd_5693_validation.xml.gz | 5.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5693 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5693 | HTTPS FTP |
-Related structure data
Related structure data | 3j46MC 5691C 5692C 3j45C 4v4nC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_5693.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | ribosome-nascent chain-SecYEG complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.12 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Segmentation: segmented large ribosomal subunit
Annotation | segmented large ribosomal subunit | ||||||||||||
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File | emd_5693_msk_1.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: segmented A and P-site tRNAs
Annotation | segmented A and P-site tRNAs | ||||||||||||
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File | emd_5693_msk_2.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: micelle map from channel density
Annotation | micelle map from channel density | ||||||||||||
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File | emd_5693_msk_3.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: zoned SecYEG channel
Annotation | zoned SecYEG channel | ||||||||||||
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File | emd_5693_msk_4.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: full channel density with micelle and nascent chain
Annotation | full channel density with micelle and nascent chain | ||||||||||||
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File | emd_5693_msk_5.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: full nascent chain density, excluding fragments in the tunnel
Annotation | full nascent chain density, excluding fragments in the tunnel | ||||||||||||
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File | emd_5693_msk_6.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: nascent chain density with signal sequence helix in the channel
Annotation | nascent chain density with signal sequence helix in the channel | ||||||||||||
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File | emd_5693_msk_7.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: segmented S1 density, excluding flexible fragments
Annotation | segmented S1 density, excluding flexible fragments | ||||||||||||
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File | emd_5693_msk_8.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: segmented small subunit
Annotation | segmented small subunit | ||||||||||||
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File | emd_5693_msk_9.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : ribosome-nascent chain-SecYEG complex
Entire | Name: ribosome-nascent chain-SecYEG complex |
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Components |
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-Supramolecule #1000: ribosome-nascent chain-SecYEG complex
Supramolecule | Name: ribosome-nascent chain-SecYEG complex / type: sample / ID: 1000 Details: Sample was prepared by crosslinking the nascent chain within stalled membrane associated ribosomes in bacteria, then purified for cryo-EM. Oligomeric state: monomer / Number unique components: 5 |
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Molecular weight | Theoretical: 2.5 MDa |
-Supramolecule #1: membrane-bound 70S ribosome
Supramolecule | Name: membrane-bound 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: EP72 / Location in cell: inner membrane |
Molecular weight | Theoretical: 2.5 MDa |
-Macromolecule #1: preprotein translocase
Macromolecule | Name: preprotein translocase / type: protein_or_peptide / ID: 1 / Name.synonym: SecYEG channel / Details: SecY: P0AGA2, SecE: P0AG96, SecG: P0AG99 / Number of copies: 1 / Oligomeric state: heterotrimer / Recombinant expression: Yes |
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Source (natural) | Organism: Escherichia coli (E. coli) / Strain: EP72 / Location in cell: inner membrane |
Molecular weight | Theoretical: 73.4 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: EP72 / Recombinant plasmid: pACYC-EhG/Y |
-Macromolecule #2: S1P
Macromolecule | Name: S1P / type: protein_or_peptide / ID: 2 Name.synonym: endogenous E. coli small ribosomal subunit protein Number of copies: 1 / Oligomeric state: monomer / Recombinant expression: No / Database: NCBI |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 61 KDa |
-Macromolecule #3: transfer RNA
Macromolecule | Name: transfer RNA / type: rna / ID: 3 / Name.synonym: tRNA Details: Both A- and P-site tRNAs present in purified ribosome-nascent chain-SecYEG complex, stalled with SecM sequence Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 25 KDa |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 8 mg/mL |
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Buffer | pH: 7.2 Details: 50 mM Tris-acetate, 10 mM Mg(OAc)2, 80 mM KOAc, 0.06% DDM |
Grid | Details: 400 mesh Quantifoil holey grids with 2/1 or 1.2/1.2 |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK III Details: A homemade freezing device with N2 gas driven plunger was also used to prepare grids in a cabinet to maintain humidity. Method: Blot 1-2 seconds before plunging. |
-Electron microscopy
Microscope | FEI TECNAI F20 |
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Temperature | Min: 94 K |
Alignment procedure | Legacy - Astigmatism: Carbon grain was imaged at ~175000 times magnification and Thon rings were optimized manually, as visualized on a Fourier transform of ccd images. |
Details | Low dose imaging: automated single particle data collection program from TVIPS was used. |
Date | Feb 10, 2012 |
Image recording | Category: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 4900 / Average electron dose: 20 e/Å2 Details: CCD 4k x 4k image frames: best 4900 from ~5900 images used for data processing. Bits/pixel: 16 |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 42000 |
Sample stage | Specimen holder: Oxford cold holder / Specimen holder model: OTHER |
Experimental equipment | Model: Tecnai F20 / Image courtesy: FEI Company |
-Image processing
Details | ~450000 particles were selected with e2boxer, used for per ccd frame CTF correction, and then subjected to unsupervised classification to remove aggregates to give 167000 particles. These particles were subjected to supervised classification in 2 steps to give a data set enriched in channels. Particles with the best signal to noise ratio were identified using the FRC comparator from the refinemulti run and classified with e2ligandclassify.py. |
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CTF correction | Details: per micrograph |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.1 Å / Resolution method: OTHER / Software - Name: EMAN2 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. Number images used: 53000 |
Final two d classification | Number classes: 5300 |
-Atomic model buiding 1
Initial model | PDB ID: 2i2p |
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Software | Name: Chimera, MDFF |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3j46: |
-Atomic model buiding 2
Initial model | PDB ID: 3j01 |
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Software | Name: Chimera, MDFF |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3j46: |
-Atomic model buiding 3
Initial model | PDB ID: 3i8g Chain - #0 - Chain ID: B / Chain - #1 - Chain ID: C |
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Software | Name: Chimera, MDFF |
Details | A- and P-site tRNAs from T. thermophilus ribosome structure. mRNA from this structure also used for some modeling steps. |
Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-3j46: |