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- EMDB-2446: Visualization of a polytopic membrane protein egressing from the ... -

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Entry
Database: EMDB / ID: EMD-2446
TitleVisualization of a polytopic membrane protein egressing from the SecY complex - Electron cryo-microscopy of an tightly coupled RNC-SecY complex
Map dataVisualization of a polytopic membrane protein egressing from the SecY complex
Sample
  • Sample: TnaC stalled E.coli ribosome in complex with SecYE
  • Complex: 70S-Ribosome
  • Protein or peptide: SecY
  • Protein or peptide: SecE
KeywordsProtein translocation / Ribosome / SecY / SecE / Proteorhodopsin
Function / homology
Function and homology information


light-activated monoatomic ion channel activity / protein insertion into membrane from inner side / cell envelope Sec protein transport complex / intracellular protein transmembrane transport / protein transport by the Sec complex / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / protein transmembrane transporter activity / photoreceptor activity / protein secretion ...light-activated monoatomic ion channel activity / protein insertion into membrane from inner side / cell envelope Sec protein transport complex / intracellular protein transmembrane transport / protein transport by the Sec complex / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / protein transmembrane transporter activity / photoreceptor activity / protein secretion / phototransduction / protein targeting / proton transmembrane transport / intracellular protein transport / membrane / plasma membrane
Similarity search - Function
Proteorhodopsin / 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 ...Proteorhodopsin / 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 / Bacterial rhodopsins signature 1. / Rhodopsin, retinal binding site / Bacteriorhodopsin-like protein / Archaeal/bacterial/fungal rhodopsins / Bacteriorhodopsin-like protein
Similarity search - Domain/homology
Protein translocase subunit SecY / Protein translocase subunit SecE / Protein translocase subunit SecY / Green-light absorbing proteorhodopsin
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.28 Å
AuthorsBischoff L / Wickles S / Berninghausen O / van der Sluis E / Beckmann R
CitationJournal: Nat Commun / Year: 2014
Title: Visualization of a polytopic membrane protein during SecY-mediated membrane insertion.
Authors: Lukas Bischoff / Stephan Wickles / Otto Berninghausen / Eli O van der Sluis / Roland Beckmann /
Abstract: The biogenesis of polytopic membrane proteins occurs co-translationally on ribosomes that are tightly bound to a membrane-embedded protein-conducting channel: the Sec-complex. The path that is ...The biogenesis of polytopic membrane proteins occurs co-translationally on ribosomes that are tightly bound to a membrane-embedded protein-conducting channel: the Sec-complex. The path that is followed by nascent proteins inside the ribosome and the Sec-complex is relatively well established; however, it is not clear what the fate of the N-terminal transmembrane domains (TMDs) of polytopic membrane proteins is when the C-terminal TMDs domains are not yet synthesized. Here, we present the sub-nanometer cryo-electron microscopy structure of an in vivo generated ribosome-SecY complex that carries a membrane insertion intermediate of proteorhodopsin (PR). The structure reveals a pre-opened Sec-complex and the first two TMDs of PR already outside the SecY complex directly in front of its proposed lateral gate. Thus, our structure is in agreement with positioning of N-terminal TMDs at the periphery of SecY, and in addition, it provides clues for the molecular mechanism underlying membrane protein topogenesis.
History
DepositionAug 26, 2013-
Header (metadata) releaseSep 25, 2013-
Map releaseJun 18, 2014-
UpdateAug 26, 2015-
Current statusAug 26, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5abb
  • Surface level: 0.2
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5abb
  • Surface level: 0.12
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5abb
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_2446.jpg

Downloads & links

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Map

FileDownload / File: emd_2446.map.gz / Format: CCP4 / Size: 185.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationVisualization of a polytopic membrane protein egressing from the SecY complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.05 Å/pix.
x 368 pix.
= 385.995 Å		1.05 Å/pix.
x 368 pix.
= 385.995 Å		1.05 Å/pix.
x 368 pix.
= 385.995 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0489 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.2 / Movie #1: 0.2
Minimum - Maximum-0.58672941 - 1.70602858
Average (Standard dev.)0.0131344 (±0.14190418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-184-184-183
Dimensions368368368
Spacing368368368
CellA=B=C: 385.9952 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.04889945652171.04889945652171.0488994565217
M x/y/z368368368
origin x/y/z0.0000.0000.000
length x/y/z385.995385.995385.995
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS-184-184-183
NC/NR/NS368368368
D min/max/mean-0.5871.7060.013

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Supplemental data

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Sample components

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Entire : TnaC stalled E.coli ribosome in complex with SecYE

EntireName: TnaC stalled E.coli ribosome in complex with SecYE
Components
  • Sample: TnaC stalled E.coli ribosome in complex with SecYE
  • Complex: 70S-Ribosome
  • Protein or peptide: SecY
  • Protein or peptide: SecE

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Supramolecule #1000: TnaC stalled E.coli ribosome in complex with SecYE

SupramoleculeName: TnaC stalled E.coli ribosome in complex with SecYE / type: sample / ID: 1000 / Number unique components: 3

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Supramolecule #1: 70S-Ribosome

SupramoleculeName: 70S-Ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli) / Strain: KC6

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Macromolecule #1: SecY

MacromoleculeName: SecY / type: protein_or_peptide / ID: 1 / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #2: SecE

MacromoleculeName: SecE / type: protein_or_peptide / ID: 2 / Recombinant expression: No
Source (natural)Organism: Escherichia coli (E. coli)

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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

BufferpH: 7.5
Details: 20 mM Tris 150 mM NH4Cl 10 mM MgCl2 0.05%DDM 125 mM Sucrose
GridDetails: The freshly prepared PR2Q-RNC-SecYEG complex was applied to 2 nm pre-coated Quantifoil R3/3 holey carbon supported grids and vitrified
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
DateMay 21, 2012
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F416 (4k x 4k) / Number real images: 16500 / Average electron dose: 20 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 148721 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 148721
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected using the automatic selection program SIGNATURE
CTF correctionDetails: Defocus group
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 7.28 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 47471

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