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- EMDB-5692: Structure of the SecY protein translocation channel in action -

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Basic information

Entry
Database: EMDB / ID: EMD-5692
TitleStructure of the SecY protein translocation channel in action
Map dataReconstruction of E. coli ribosome-SecYEG complex
Sample
  • Sample: E. coli 70S ribosome with recombinant E. coli SecYEG
  • Complex: non-translating 70S ribosome
  • Protein or peptide: SecYEGSec61
Keywordsribosome-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
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 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 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: 9.5 Å
AuthorsPark P / 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 14, 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: 1.8
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j45
  • Surface level: 1.8
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j45
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5692.jpg

Downloads & links

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Map

FileDownload / File: emd_5692.map.gz / Format: CCP4 / Size: 11.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of E. coli ribosome-SecYEG complex
Voxel sizeX=Y=Z: 2.73 Å
Density
Contour LevelBy AUTHOR: 1.8 / Movie #1: 1.8
Minimum - Maximum-5.4818368 - 11.23235416
Average (Standard dev.)0.20448679 (±0.92750901)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-73-73-73
Dimensions144144144
Spacing144144144
CellA=B=C: 393.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.732.732.73
M x/y/z144144144
origin x/y/z0.0000.0000.000
length x/y/z393.120393.120393.120
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-73-73-73
NC/NR/NS144144144
D min/max/mean-5.48211.2320.204

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Supplemental data

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Segmentation: large ribosomal subunit sub-volume

Annotationlarge ribosomal subunit sub-volume
Fileemd_5692_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: full channel sub-volume

Annotationfull channel sub-volume
Fileemd_5692_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: zoned SecYEG sub-volume

Annotationzoned SecYEG sub-volume
Fileemd_5692_msk_3.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: Zoned micelle sub-volume

AnnotationZoned micelle sub-volume
Fileemd_5692_msk_4.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: small ribosomal subunit sub-volume

Annotationsmall ribosomal subunit sub-volume
Fileemd_5692_msk_5.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 : E. coli 70S ribosome with recombinant E. coli SecYEG

EntireName: E. coli 70S ribosome with recombinant E. coli SecYEG
Components
  • Sample: E. coli 70S ribosome with recombinant E. coli SecYEG
  • Complex: non-translating 70S ribosome
  • Protein or peptide: SecYEGSec61

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Supramolecule #1000: E. coli 70S ribosome with recombinant E. coli SecYEG

SupramoleculeName: 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
Molecular weightTheoretical: 2.5 MDa

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Supramolecule #1: non-translating 70S ribosome

SupramoleculeName: non-translating 70S ribosome / type: complex / ID: 1 / Recombinant expression: No / Database: NCBI / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: MRE600 / Location in cell: cytoplasm
Molecular weightTheoretical: 2.4 MDa

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Macromolecule #1: SecYEG

MacromoleculeName: 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
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: inner membrane
Molecular weightTheoretical: 73.4 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: C43(DE3) / Recombinant plasmid: pBAD-EhisYG

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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

Concentration2 mg/mL
BufferpH: 7.5
Details: 50 mM HEPES-KOH, 100 mM KOAc, 10 mM Mg(OAc)2, 0.05% DDM
GridDetails: 400 mesh Cu grids with continuous or holey carbon films
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER / Method: Blot 1 second before plunging.

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 51000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 50000
Sample stageSpecimen holder: 626 single tilt / Specimen holder model: GATAN LIQUID NITROGEN / Tilt angle max: 30
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: imaging of carbon film at 175,000 times magnification
Detailslow dose imaging with manual data collection
DateApr 10, 2006
Image recordingCategory: 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 min0

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Image processing

CTF correctionDetails: per micrograph
Final two d classificationNumber classes: 1900
Final reconstructionAlgorithm: 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
DetailsParticles were picked with boxer and CTF-corrected with EMAN1.

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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-3j45:
Structure of a non-translocating SecY protein channel with the 70S ribosome

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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-3j45:
Structure of a non-translocating SecY protein channel with the 70S ribosome

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