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- EMDB-2565: Cryo-EM of SecA-70S complex -

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Basic information

Entry
Database: EMDB / ID: EMD-2565
TitleCryo-EM of SecA-70S complex
Map datareconstruction of monomeric SecA bound to 70S ribosome
Sample
  • Sample: SecA-70S ribosome
  • Complex: 70S ribosomeRibosome
  • Complex: SecA
KeywordsSecA / ribosome
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 10.3 Å
AuthorsSingh R / Kraft C / Jaiswal R / Sejwal K / Kasaragod V / Kuper J / Buerger J / Mielke T / Luirink J / Bhushan S
CitationJournal: J Biol Chem / Year: 2014
Title: Cryo-electron microscopic structure of SecA protein bound to the 70S ribosome.
Authors: Rajkumar Singh / Christian Kraft / Rahul Jaiswal / Kushal Sejwal / Vikram Babu Kasaragod / Jochen Kuper / Jörg Bürger / Thorsten Mielke / Joen Luirink / Shashi Bhushan /
Abstract: SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the ...SecA is an ATP-dependent molecular motor pumping secretory and outer membrane proteins across the cytoplasmic membrane in bacteria. SecA associates with the protein-conducting channel, the heterotrimeric SecYEG complex, in a so-called posttranslational manner. A recent study further showed binding of a monomeric state of SecA to the ribosome. However, the true oligomeric state of SecA remains controversial because SecA can also form functional dimers, and high-resolution crystal structures exist for both the monomer and the dimer. Here we present the cryo-electron microscopy structures of Escherichia coli SecA bound to the ribosome. We show that not only a monomeric SecA binds to the ribosome but also that two copies of SecA can be observed that form an elongated dimer. Two copies of SecA completely surround the tunnel exit, providing a unique environment to the nascent polypeptides emerging from the ribosome. We identified the N-terminal helix of SecA required for a stable association with the ribosome. The structures indicate a possible function of the dimeric form of SecA at the ribosome.
History
DepositionJan 20, 2014-
Header (metadata) releaseMar 12, 2014-
Map releaseMar 12, 2014-
UpdateMay 21, 2014-
Current statusMay 21, 2014Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 58.7
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 58.7
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-2565-dim...

Downloads & links

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Map

FileDownload / File: emd_2565.map.gz / Format: CCP4 / Size: 94.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationreconstruction of monomeric SecA bound to 70S ribosome
Voxel sizeX=Y=Z: 1.24 Å
Density
Contour LevelBy AUTHOR: 58.700000000000003 / Movie #1: 58.7
Minimum - Maximum-280.310943599999973 - 672.746276859999966
Average (Standard dev.)20.007276539999999 (±74.508453369999998)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-147-147-146
Dimensions294294294
Spacing294294294
CellA=B=C: 364.56 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.241.241.24
M x/y/z294294294
origin x/y/z0.0000.0000.000
length x/y/z364.560364.560364.560
α/β/γ90.00090.00090.000
start NX/NY/NZ-207-207-206
NX/NY/NZ414414414
MAP C/R/S123
start NC/NR/NS-147-147-146
NC/NR/NS294294294
D min/max/mean-280.311672.74620.007

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Supplemental data

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Sample components

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Entire : SecA-70S ribosome

EntireName: SecA-70S ribosome
Components
  • Sample: SecA-70S ribosome
  • Complex: 70S ribosomeRibosome
  • Complex: SecA

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Supramolecule #1000: SecA-70S ribosome

SupramoleculeName: SecA-70S ribosome / type: sample / ID: 1000 / Oligomeric state: Monomeric SecA bound to the 70S ribosome / Number unique components: 2
Molecular weightExperimental: 2 MDa / Theoretical: 2 MDa

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Supramolecule #1: 70S ribosome

SupramoleculeName: 70S ribosome / type: complex / ID: 1 / Name.synonym: 70S / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 2 MDa / Theoretical: 2 MDa

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Supramolecule #2: SecA

SupramoleculeName: SecA / type: complex / ID: 2 / Name.synonym: SecA / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 100 KDa / Theoretical: 100 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

BufferpH: 7.6
Details: 40 mM Hepes pH 7.6, 50 mM K-acetate, 25 mM Mg-Acetate, 5mM DTT, 0.1% protease inhibitor pill/ml, 0.1 U/ml RNAsin, 125 mM sucrose
GridDetails: 2-nm carbon-coated holey grids
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III / Method: Blot for 5 seconds before plunging

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 39000
Sample stageSpecimen holder model: GATAN LIQUID NITROGEN
DateJun 11, 2011
Image recordingCategory: FILM / Film or detector model: KODAK 4489 FILM / Digitization - Scanner: OTHER / Digitization - Sampling interval: 0.25 µm / Number real images: 300 / Average electron dose: 25 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each Micrographs
Final angle assignmentDetails: Spider
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 10.3 Å / Resolution method: OTHER / Software - Name: Spider / Number images used: 83000
DetailsSpider

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Atomic model buiding 1

Initial modelPDB ID:

1m74

SoftwareName: Chimera
DetailsThe domains were separately fitted by manual docking using coot and chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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