+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-5692 | |||||||||
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Title | Structure of the SecY protein translocation channel in action | |||||||||
Map data | Reconstruction of E. coli ribosome-SecYEG complex | |||||||||
Sample |
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Keywords | ribosome-channel complex / co-translational translocation / SecYEG | |||||||||
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 / intracellular protein transport ...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 / intracellular protein transport / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / 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: 9.5 Å | |||||||||
Authors | Park P / 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_5692.map.gz | 1.4 MB | EMDB map data format | |
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Header (meta data) | emd-5692-v30.xml emd-5692.xml | 23.9 KB 23.9 KB | Display Display | EMDB header |
Images | emd_5692.jpg | 164.1 KB | ||
Masks | emd_5692_msk_1.map emd_5692_msk_2.map emd_5692_msk_3.map emd_5692_msk_4.map emd_5692_msk_5.map | 11.4 MB 11.4 MB 113.6 KB 11.4 MB 11.4 MB | Mask map | |
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-5692 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-5692 | HTTPS FTP |
-Validation report
Summary document | emd_5692_validation.pdf.gz | 488.6 KB | Display | EMDB validaton report |
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Full document | emd_5692_full_validation.pdf.gz | 488.2 KB | Display | |
Data in XML | emd_5692_validation.xml.gz | 5.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5692 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-5692 | HTTPS FTP |
-Related structure data
Related structure data | 3j45MC 5691C 5693C 3j46C 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_5692.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Reconstruction of E. coli ribosome-SecYEG complex | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 2.73 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Segmentation: large ribosomal subunit sub-volume
Annotation | large ribosomal subunit sub-volume | ||||||||||||
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File | emd_5692_msk_1.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: full channel sub-volume
Annotation | full channel sub-volume | ||||||||||||
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File | emd_5692_msk_2.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: zoned SecYEG sub-volume
Annotation | zoned SecYEG sub-volume | ||||||||||||
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File | emd_5692_msk_3.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: Zoned micelle sub-volume
Annotation | Zoned micelle sub-volume | ||||||||||||
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File | emd_5692_msk_4.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Segmentation: small ribosomal subunit sub-volume
Annotation | small ribosomal subunit sub-volume | ||||||||||||
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File | emd_5692_msk_5.map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : E. coli 70S ribosome with recombinant E. coli SecYEG
Entire | Name: E. coli 70S ribosome with recombinant E. coli SecYEG |
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Components |
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-Supramolecule #1000: E. coli 70S ribosome with recombinant E. coli SecYEG
Supramolecule | Name: E. coli 70S ribosome with recombinant E. coli SecYEG / type: sample / ID: 1000 Details: Ribosome-SecY complexes were prepared by mixing ribosomes at 4 uM with SecY (32 uM) and incubating them on ice for 30 min before freezing. Oligomeric state: one ribosome and one SecYEG / Number unique components: 2 |
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Molecular weight | Theoretical: 2.5 MDa |
-Supramolecule #1: non-translating 70S ribosome
Supramolecule | Name: non-translating 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: MRE600 / Location in cell: cytoplasm |
Molecular weight | Theoretical: 2.4 MDa |
-Macromolecule #1: SecYEG
Macromolecule | Name: SecYEG / type: protein_or_peptide / ID: 1 / Name.synonym: Sec translocase / 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) / Location in cell: inner membrane |
Molecular weight | Theoretical: 73.4 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: C43(DE3) / Recombinant plasmid: pBAD-EhisYG |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 2 mg/mL |
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Buffer | pH: 7.5 Details: 50 mM HEPES-KOH, 100 mM KOAc, 10 mM Mg(OAc)2, 0.05% DDM |
Grid | Details: 400 mesh Cu grids with continuous or holey carbon films |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER / Method: Blot 1 second before plunging. |
-Electron microscopy
Microscope | FEI TECNAI 20 |
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Temperature | Average: 93 K |
Alignment procedure | Legacy - Astigmatism: imaging of carbon film at 175,000 times magnification |
Details | low dose imaging with manual data collection |
Date | Apr 10, 2006 |
Image recording | Category: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 4.5 µm / Number real images: 360 / Average electron dose: 20 e/Å2 / Od range: 1 / Bits/pixel: 16 |
Tilt angle min | 0 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 51000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000 |
Sample stage | Specimen holder: 626 single tilt / Specimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 30 |
-Image processing
Details | Particles were picked with boxer and CTF-corrected with EMAN1. |
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CTF correction | Details: per micrograph |
Final reconstruction | Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.5 Å / Resolution method: OTHER / Software - Name: EMAN1 Details: CTF correction was done on untilted and 30 degree tilted images. Number images used: 39000 |
Final two d classification | Number classes: 1900 |
-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-3j45: |