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- EMDB-1829: SecM-stalled ribosomes adopt an altered geometry at the peptidylt... -

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Basic information

Entry
Database: EMDB / ID: EMD-1829
TitleSecM-stalled ribosomes adopt an altered geometry at the peptidyltransferase center
Map dataSecM stalled ribosome
Sample
  • Sample: SecM-stalled RNC
  • Complex: 70S
KeywordsSecM-stalled RNC
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 5.6 Å
AuthorsBhushan S / Hoffman T / Seidelt B / Frauenfeld J / Mielke T / Berninghausen O / Wilson D / Beckmann R
CitationJournal: PLoS Biol / Year: 2011
Title: SecM-stalled ribosomes adopt an altered geometry at the peptidyl transferase center.
Authors: Shashi Bhushan / Thomas Hoffmann / Birgit Seidelt / Jens Frauenfeld / Thorsten Mielke / Otto Berninghausen / Daniel N Wilson / Roland Beckmann /
Abstract: As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce ...As nascent polypeptide chains are synthesized, they pass through a tunnel in the large ribosomal subunit. Interaction between specific nascent chains and the ribosomal tunnel is used to induce translational stalling for the regulation of gene expression. One well-characterized example is the Escherichia coli SecM (secretion monitor) gene product, which induces stalling to up-regulate translation initiation of the downstream secA gene, which is needed for protein export. Although many of the key components of SecM and the ribosomal tunnel have been identified, understanding of the mechanism by which the peptidyl transferase center of the ribosome is inactivated has been lacking. Here we present a cryo-electron microscopy reconstruction of a SecM-stalled ribosome nascent chain complex at 5.6 Å. While no cascade of rRNA conformational changes is evident, this structure reveals the direct interaction between critical residues of SecM and the ribosomal tunnel. Moreover, a shift in the position of the tRNA-nascent peptide linkage of the SecM-tRNA provides a rationale for peptidyl transferase center silencing, conditional on the simultaneous presence of a Pro-tRNA(Pro) in the ribosomal A-site. These results suggest a distinct allosteric mechanism of regulating translational elongation by the SecM stalling peptide.
History
DepositionNov 22, 2010-
Header (metadata) releaseNov 26, 2010-
Map releaseJun 22, 2011-
UpdateOct 10, 2012-
Current statusOct 10, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.188
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.188
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1829.tif

Downloads & links

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Map

FileDownload / File: emd_1829.map.gz / Format: CCP4 / Size: 94.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSecM stalled ribosome
Voxel sizeX=Y=Z: 1.237 Å
Density
Contour LevelBy AUTHOR: 0.188 / Movie #1: 0.188
Minimum - Maximum-0.780446 - 1.90893
Average (Standard dev.)0.0108189 (±0.123236)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-147-147-146
Dimensions294294294
Spacing294294294
CellA=B=C: 363.678 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.2371.2371.237
M x/y/z294294294
origin x/y/z0.0000.0000.000
length x/y/z363.678363.678363.678
α/β/γ90.00090.00090.000
start NX/NY/NZ-147-147-146
NX/NY/NZ294294294
MAP C/R/S213
start NC/NR/NS-147-147-146
NC/NR/NS294294294
D min/max/mean-0.7801.9090.011

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Supplemental data

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Sample components

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Entire : SecM-stalled RNC

EntireName: SecM-stalled RNC
Components
  • Sample: SecM-stalled RNC
  • Complex: 70S

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Supramolecule #1000: SecM-stalled RNC

SupramoleculeName: SecM-stalled RNC / type: sample / ID: 1000 / Details: no / Number unique components: 1
Molecular weightExperimental: 2.3 MDa / Theoretical: 2.3 MDa

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Supramolecule #1: 70S

SupramoleculeName: 70S / type: complex / ID: 1 / Name.synonym: 70S / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: ALL
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightExperimental: 2.3 MDa / Theoretical: 2.3 MDa

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7
Details: 20 mM Hepes Ph 7.0, 50 mM Pot Acetate, 6 mM Mag Acetate, 5 mM DTT, 500 ug/ml Chloroamphenicol, 0.05 % Nikkol, 0.5 % pill/ml, 0.1 U/ml RNAsin, 125 mM sucrose
StainingType: NEGATIVE / Details: native
GridDetails: 2 nM carbon coated 200 mesh Cu grid
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot / Method: 10 seconds for blotting

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 4.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 38900
Specialist opticsEnergy filter - Name: FEI
Sample stageSpecimen holder: Eucentric / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: PRIMESCAN / Number real images: 400 / Average electron dose: 25 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: CTFFIND on Micrographs
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 5.6 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: Spider / Number images used: 544000
DetailsProcessed using Spider

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Atomic model buiding 1

Initial modelPDB ID:

2wwq
PDB Unreleased entry

SoftwareName: Chimera
DetailsFitted manually using Chimera and Coot
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

2wwl
PDB Unreleased entry

SoftwareName: Chimera
DetailsFitted manually using Chimera and Coot
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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