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- EMDB-5691: Structure of the SecY protein translocation channel in action -

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Basic information

Entry
Database: EMDB / ID: EMD-5691
TitleStructure of the SecY protein translocation channel in action
Map dataReconstruction of archaeal 70S ribosome-SecYEbeta complex from Methanococcus jannaschii
Sample
  • Sample: Methanococcus jannaschii 70S ribosome-SecYEbeta complex
  • Complex: non-translating 70S ribosome
  • Protein or peptide: SecYEbeta
  • RNA: transfer RNA
Keywordsarchaeal 70S ribosome / SecYEbeta channel / co-translational translocation
Function / homology
Function and homology information


intracellular protein transmembrane transport / SRP-dependent cotranslational protein targeting to membrane, translocation / signal sequence binding / protein secretion / protein transmembrane transporter activity / protein targeting / protein transport / plasma membrane
Similarity search - Function
Preprotein translocase subunit SecG / Protein translocase subunit SecY / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / Protein secY signature 1. / Protein secY signature 2. ...Preprotein translocase subunit SecG / Protein translocase subunit SecY / Protein transport protein SecG/Sec61-beta/Sbh / Sec61beta family / Protein translocase SEC61 complex, gamma subunit / Protein translocase SecE domain superfamily / Translocon Sec61/SecY, plug domain / Plug domain of Sec61p / 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
Similarity search - Domain/homology
Preprotein translocase subunit SecG / Protein translocase subunit SecE / Protein translocase subunit SecY
Similarity search - Component
Biological speciesMethanocaldococcus jannaschii (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.0 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-
UpdateJul 23, 2014-
Current statusJul 23, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.93
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.93
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-4v4n
  • Surface level: 0.93
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5691.jpg

Downloads & links

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Map

FileDownload / File: emd_5691.map.gz / Format: CCP4 / Size: 16 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of archaeal 70S ribosome-SecYEbeta complex from Methanococcus jannaschii
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.73 Å/pix.
x 162 pix.
= 442.26 Å		2.73 Å/pix.
x 163 pix.
= 444.99 Å		2.73 Å/pix.
x 163 pix.
= 444.99 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 2.73 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.93 / Movie #1: 0.93
Minimum - Maximum-0.75200582 - 3.55626702
Average (Standard dev.)0.05997659 (±0.26984879)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-81-81-81
Dimensions163163162
Spacing163163162
CellA: 444.99 Å / B: 444.99 Å / C: 442.26 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.732.732.73
M x/y/z163163162
origin x/y/z0.0000.0000.000
length x/y/z444.990444.990442.260
α/β/γ90.00090.00090.000
start NX/NY/NZ-132-122-147
NX/NY/NZ250274261
MAP C/R/S123
start NC/NR/NS-81-81-81
NC/NR/NS163163162
D min/max/mean-0.7523.5560.060

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Supplemental data

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Segmentation: zone large ribosomal subunit

Annotationzone large ribosomal subunit
Fileemd_5691_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: zoned full channel desnity

Annotationzoned full channel desnity
Fileemd_5691_msk_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: segmented density with only SecYEbeta

Annotationsegmented density with only SecYEbeta
Fileemd_5691_msk_3.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: zoned micelle

Annotationzoned micelle
Fileemd_5691_msk_4.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Segmentation: zoned small ribosomal subunit

Annotationzoned small ribosomal subunit
Fileemd_5691_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 : Methanococcus jannaschii 70S ribosome-SecYEbeta complex

EntireName: Methanococcus jannaschii 70S ribosome-SecYEbeta complex
Components
  • Sample: Methanococcus jannaschii 70S ribosome-SecYEbeta complex
  • Complex: non-translating 70S ribosome
  • Protein or peptide: SecYEbeta
  • RNA: transfer RNA

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Supramolecule #1000: Methanococcus jannaschii 70S ribosome-SecYEbeta complex

SupramoleculeName: Methanococcus jannaschii 70S ribosome-SecYEbeta complex
type: sample / ID: 1000
Details: The sample was reconstituted from ribosomal subunits purified from M. jannaschii and from recombinant SecYEbeta.
Oligomeric state: one 70S, one E-site tRNA, one SecYEbeta channel
Number unique components: 3
Molecular weightTheoretical: 2.6 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: Methanocaldococcus jannaschii (archaea) / Location in cell: cytoplasm
Molecular weightTheoretical: 2.5 MDa

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Macromolecule #1: SecYEbeta

MacromoleculeName: SecYEbeta / type: protein_or_peptide / ID: 1 / Name.synonym: SecY channel / Details: SecY: Q60175.2, SecE: Q57817.1, Secbeta: P60460.1 / Number of copies: 1 / Oligomeric state: heterotrimer / Recombinant expression: Yes
Source (natural)Organism: Methanocaldococcus jannaschii (archaea) / Location in cell: inner membrane
Molecular weightTheoretical: 61.8 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: C43(DE3) / Recombinant plasmid: pBAD22

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Macromolecule #2: transfer RNA

MacromoleculeName: transfer RNA / type: rna / ID: 2 / Name.synonym: tRNA / Details: co-purified with the ribosomal subunits / Classification: TRANSFER / Structure: DOUBLE HELIX / Synthetic?: No
Source (natural)Organism: Methanocaldococcus jannaschii (archaea)
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

Concentration2 mg/mL
BufferpH: 7.5
Details: 100 mM NH4Cl, 30 mM MgCl2, 20 mM HEPES-KOH, 6 mM beta-mercaptoethanol, 0.1% DDM
GridDetails: Quantifoil 400 mesh 2/1 Cu grids, air glow discharged, and 400 mesh Cu grids with a thin continuous carbon foil
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 77 K / Instrument: FEI VITROBOT MARK III / Method: Blot 1-2 seconds before plunging.

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Electron microscopy

MicroscopeFEI TECNAI F20
TemperatureAverage: 93 K
Alignment procedureLegacy - Astigmatism: corrected at 175,000 times magnification
DetailsLow dose imaging, data collected manually
DateApr 10, 2008
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: OTHER / Number real images: 217 / Average electron dose: 20 e/Å2 / Details: Zeiss SCAI scanner was also used. / Od range: 1 / Bits/pixel: 16
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated 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 stageSpecimen holder: Oxford holder / Specimen holder model: GATAN LIQUID NITROGEN
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

DetailsParticles were selected with e2boxer and CTF-corrected with EMAN2. Final maps were obtained from 6 different refinement conditions, aligned in Chimera, and averaged to produce the final map.
CTF correctionDetails: per micrograph
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.0 Å / Resolution method: OTHER / Software - Name: EMAN1, EMAN2
Details: Final map calculated as an average of 6 different refinements in EMAN2 with different parameters.
Number images used: 37000
Final two d classificationNumber classes: 3800

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Atomic model buiding 1

Initial modelPDB ID:

3j21
PDB Unreleased entry

SoftwareName: Chimera, MDFF
DetailsDocked with Chimera and fit with MDFF. Chains omitted: 1('el' 1-77), 6(L83-1), 4(L83-2), c(L33e). Chains truncated: a(L31e) 78-85, W(L29) 67-72, C(L3) 103-125.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4v4n:
Structure of the Methanococcus jannaschii ribosome-SecYEBeta channel complex

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Atomic model buiding 2

Initial modelPDB ID:

3j2l
PDB Unreleased entry

SoftwareName: Chimera, MDFF
DetailsRegions omitted: rRNA chain 1 [1-6, 3047-3049 5' and 3' ends 23S], 150-163, 750-756, 1311-1321, 2904-2942 (ES).
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4v4n:
Structure of the Methanococcus jannaschii ribosome-SecYEBeta channel complex

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Atomic model buiding 3

Initial modelPDB ID:

3j20
PDB Unreleased entry

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

PDB-4v4n:
Structure of the Methanococcus jannaschii ribosome-SecYEBeta channel complex

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Atomic model buiding 4

Initial modelPDB ID:

1rhz


Chain - #0 - Chain ID: A / Chain - #1 - Chain ID: B / Chain - #2 - Chain ID: C
SoftwareName: Chimera, MDFF
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-4v4n:
Structure of the Methanococcus jannaschii ribosome-SecYEBeta channel complex

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