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- EMDB-5693: Structure of the SecY protein translocation channel in action -

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Basic information

Entry
Database: EMDB / ID: EMD-5693
TitleStructure of the SecY protein translocation channel in action
Map dataribosome-nascent chain-SecYEG complex
Sample
  • Sample: ribosome-nascent chain-SecYEG complex
  • Complex: membrane-bound 70S ribosome
  • Protein or peptide: preprotein translocase
  • Protein or peptide: S1P
  • RNA: transfer RNA
Keywordsribosome-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
Preprotein translocase SecG subunit / Preprotein translocase SecG subunit / SecE subunit of protein translocation complex, bacterial-like / SecE superfamily / Protein translocase subunit SecY / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. / SecE/Sec61-gamma subunits of protein translocation complex / Protein translocase complex, SecE/Sec61-gamma subunit ...Preprotein translocase SecG subunit / Preprotein translocase SecG subunit / SecE subunit of protein translocation complex, bacterial-like / SecE superfamily / Protein translocase subunit SecY / Protein secE/sec61-gamma signature. / Protein secY signature 1. / Protein secY signature 2. / 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 L1, bacterial-type / Ribosomal protein L1, conserved site / Ribosomal protein L1 / Ribosomal protein L1 signature. / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / Ribosomal proteins 50S L24/mitochondrial 39S L24 / Ribosomal protein L24 / Ribosomal protein L23/L25, conserved site / Ribosomal protein L23 signature. / Ribosomal protein L29, conserved site / Ribosomal protein L29 signature. / Ribosomal protein L29/L35 / Ribosomal protein L29/L35 superfamily / Ribosomal L29 protein / Ribosomal protein L24/L26, conserved site / KOW (Kyprides, Ouzounis, Woese) motif. / Ribosomal protein L25/L23 / Ribosomal protein L23 / Ribosomal protein L26/L24, KOW domain / Ribosomal protein L24 signature. / Translation protein SH3-like domain superfamily / Ribosomal protein L23/L15e core domain superfamily / KOW / KOW motif / Ribosomal protein L2, domain 2 / Nucleotide-binding alpha-beta plait domain superfamily
Similarity search - Domain/homology
Large ribosomal subunit protein uL1 / Large ribosomal subunit protein uL29 / Large ribosomal subunit protein uL23 / Protein translocase subunit SecE / Protein-export membrane protein SecG / Protein translocase subunit SecY / Large ribosomal subunit protein uL24
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.1 Å
AuthorsPark E / Menetret JF / Gumbart JC / Ludtke SJ / Li W / Whynot A / Rapoport TA / Akey CW
CitationJournal: 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
DepositionJun 18, 2013-
Header (metadata) releaseAug 21, 2013-
Map releaseOct 23, 2013-
UpdateFeb 5, 2014-
Current statusFeb 5, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j46
  • Surface level: 0.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j46
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5693_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5693.map.gz / Format: CCP4 / Size: 26.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationribosome-nascent chain-SecYEG complex
Voxel sizeX=Y=Z: 2.12 Å
Density
Contour LevelBy AUTHOR: 0.8 / Movie #1: 0.8
Minimum - Maximum-0.46406972 - 3.24496651
Average (Standard dev.)0.07153273 (±0.29414058)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-93-98-100
Dimensions192192192
Spacing192192192
CellA=B=C: 407.03998 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.122.122.12
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z407.040407.040407.040
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-98-93-100
NC/NR/NS192192192
D min/max/mean-0.4643.2450.072

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Supplemental data

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Segmentation: segmented large ribosomal subunit

Annotationsegmented large ribosomal subunit
Fileemd_5693_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: segmented A and P-site tRNAs

Annotationsegmented A and P-site tRNAs
Fileemd_5693_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: micelle map from channel density

Annotationmicelle map from channel density
Fileemd_5693_msk_3.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: zoned SecYEG channel

Annotationzoned SecYEG channel
Fileemd_5693_msk_4.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: full channel density with micelle and nascent chain

Annotationfull channel density with micelle and nascent chain
Fileemd_5693_msk_5.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: full nascent chain density, excluding fragments in the tunnel

Annotationfull nascent chain density, excluding fragments in the tunnel
Fileemd_5693_msk_6.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: nascent chain density with signal sequence helix in the channel

Annotationnascent chain density with signal sequence helix in the channel
Fileemd_5693_msk_7.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: segmented S1 density, excluding flexible fragments

Annotationsegmented S1 density, excluding flexible fragments
Fileemd_5693_msk_8.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: segmented small subunit

Annotationsegmented small subunit
Fileemd_5693_msk_9.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ribosome-nascent chain-SecYEG complex

EntireName: ribosome-nascent chain-SecYEG complex
Components
  • Sample: ribosome-nascent chain-SecYEG complex
  • Complex: membrane-bound 70S ribosome
  • Protein or peptide: preprotein translocase
  • Protein or peptide: S1P
  • RNA: transfer RNA

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Supramolecule #1000: ribosome-nascent chain-SecYEG complex

SupramoleculeName: 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
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: membrane-bound 70S ribosome

SupramoleculeName: membrane-bound 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: EP72 / Location in cell: inner membrane
Molecular weightTheoretical: 2.5 MDa

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Macromolecule #1: preprotein translocase

MacromoleculeName: 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
Source (natural)Organism: Escherichia coli (E. coli) / Strain: EP72 / Location in cell: inner membrane
Molecular weightTheoretical: 73.4 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: EP72 / Recombinant plasmid: pACYC-EhG/Y

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Macromolecule #2: S1P

MacromoleculeName: 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
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 61 KDa

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Macromolecule #3: transfer RNA

MacromoleculeName: 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
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 25 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration8 mg/mL
BufferpH: 7.2
Details: 50 mM Tris-acetate, 10 mM Mg(OAc)2, 80 mM KOAc, 0.06% DDM
GridDetails: 400 mesh Quantifoil holey grids with 2/1 or 1.2/1.2
VitrificationCryogen 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.

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 160 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 42000
Sample stageSpecimen holder: Oxford cold holder / Specimen holder model: OTHER
TemperatureMin: 94 K
Alignment procedureLegacy - Astigmatism: Carbon grain was imaged at ~175000 times magnification and Thon rings were optimized manually, as visualized on a Fourier transform of ccd images.
DetailsLow dose imaging: automated single particle data collection program from TVIPS was used.
DateFeb 10, 2012
Image recordingCategory: 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 min0
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per micrograph
Final two d classificationNumber classes: 5300
Final reconstructionAlgorithm: 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
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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Atomic model buiding 1

Initial modelPDB ID:

2i2p
PDB Unreleased entry

SoftwareName: Chimera, MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j46:
Structure of the SecY protein translocation channel in action

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Atomic model buiding 2

Initial modelPDB ID:

3j01
PDB Unreleased entry

SoftwareName: Chimera, MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j46:
Structure of the SecY protein translocation channel in action

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Atomic model buiding 3

Initial modelPDB ID:

3i8g
PDB Unreleased entry


Chain - #0 - Chain ID: B / Chain - #1 - Chain ID: C
SoftwareName: Chimera, MDFF
DetailsA- and P-site tRNAs from T. thermophilus ribosome structure. mRNA from this structure also used for some modeling steps.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-3j46:
Structure of the SecY protein translocation channel in action

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