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- EMDB-1796: Proteomic characterization of archaeal ribosomes reveals the pres... -

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Entry
Database: EMDB / ID: EMD-1796
TitleProteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins
Map dataThis is a 50S subunit of P.aerophilum Ribosome
Sample
  • Sample: Pyrobaculum aerophilum 50S ribosomal subunit
  • Complex: Pyrobaculum aerophilum 50S ribosomal subunit
Keywordsarchaea / ribosome / 50S / translation / ribosomal proteins
Biological speciesPyrobaculum aerophilum (archaea)
Methodsingle particle reconstruction / cryo EM / negative staining / Resolution: 25.0 Å
AuthorsMarquez V / Froehlich T / Armache J-P / Sohmen D / Doenhoefer A / Berninghausen O / Thomm M / Beckmann R / Arnold GJ / Wilson DN
CitationJournal: J Mol Biol / Year: 2011
Title: Proteomic characterization of archaeal ribosomes reveals the presence of novel archaeal-specific ribosomal proteins.
Authors: Viter Márquez / Thomas Fröhlich / Jean-Paul Armache / Daniel Sohmen / Alexandra Dönhöfer / Aleksandra Mikolajka / Otto Berninghausen / Michael Thomm / Roland Beckmann / Georg J Arnold / Daniel N Wilson /
Abstract: Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been ...Protein synthesis occurs in macromolecular particles called ribosomes. All ribosomes are composed of RNA and proteins. While the protein composition of bacterial and eukaryotic ribosomes has been well-characterized, a systematic analysis of archaeal ribosomes has been lacking. Here we report the first comprehensive two-dimensional PAGE and mass spectrometry analysis of archaeal ribosomes isolated from the thermophilic Pyrobaculum aerophilum and the thermoacidophilic Sulfolobus acidocaldarius Crenarchaeota. Our analysis identified all 66 ribosomal proteins (r-proteins) of the P. aerophilum small and large subunits, as well as all but two (62 of 64; 97%) r-proteins of the S. acidocaldarius small and large subunits that are predicted genomically. Some r-proteins were identified with one or two lysine methylations and N-terminal acetylations. In addition, we identify three hypothetical proteins that appear to be bona fide r-proteins of the S. acidocaldarius large subunit. Dissociation of r-proteins from the S. acidocaldarius large subunit indicates that the novel r-proteins establish tighter interactions with the large subunit than some integral r-proteins. Furthermore, cryo electron microscopy reconstructions of the S. acidocaldarius and P. aerophilum 50S subunits allow for a tentative localization of the binding site of the novel r-proteins. This study illustrates not only the potential diversity of the archaeal ribosomes but also the necessity to experimentally analyze the archaeal ribosomes to ascertain their protein composition. The discovery of novel archaeal r-proteins and factors may be the first step to understanding how archaeal ribosomes cope with extreme environmental conditions.
History
DepositionSep 28, 2010-
Header (metadata) releaseApr 8, 2011-
Map releaseApr 8, 2011-
UpdateOct 24, 2012-
Current statusOct 24, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.55
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.55
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd1796.png

Downloads & links

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Map

FileDownload / File: emd_1796.map.gz / Format: CCP4 / Size: 9.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis is a 50S subunit of P.aerophilum Ribosome
Voxel sizeX=Y=Z: 3.308 Å
Density
Contour LevelBy AUTHOR: 0.55 / Movie #1: 0.55
Minimum - Maximum-1.59045 - 3.89016
Average (Standard dev.)0.0241512 (±0.428755)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin-69-69-68
Dimensions138138138
Spacing138138138
CellA=B=C: 456.504 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.3083.3083.308
M x/y/z138138138
origin x/y/z0.0000.0000.000
length x/y/z456.504456.504456.504
α/β/γ90.00090.00090.000
start NX/NY/NZ-69-69-68
NX/NY/NZ138138138
MAP C/R/S213
start NC/NR/NS-69-69-68
NC/NR/NS138138138
D min/max/mean-1.5903.8900.024

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Supplemental data

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Sample components

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Entire : Pyrobaculum aerophilum 50S ribosomal subunit

EntireName: Pyrobaculum aerophilum 50S ribosomal subunit
Components
  • Sample: Pyrobaculum aerophilum 50S ribosomal subunit
  • Complex: Pyrobaculum aerophilum 50S ribosomal subunit

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Supramolecule #1000: Pyrobaculum aerophilum 50S ribosomal subunit

SupramoleculeName: Pyrobaculum aerophilum 50S ribosomal subunit / type: sample / ID: 1000 / Oligomeric state: One ribosomal subunit / Number unique components: 1
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 MDa / Method: Sedimentation

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Supramolecule #1: Pyrobaculum aerophilum 50S ribosomal subunit

SupramoleculeName: Pyrobaculum aerophilum 50S ribosomal subunit / type: complex / ID: 1 / Name.synonym: 50S of P.aerophilum / Recombinant expression: No / Ribosome-details: ribosome-prokaryote: LSU 50S
Source (natural)Organism: Pyrobaculum aerophilum (archaea)
Molecular weightExperimental: 1.5 MDa / Theoretical: 1.5 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: 7.5
Details: 20 mM Hepes pH 7.5, 10 mM Mg(OAc)2, 30 mM NH4OAc, 4 mM beta-Mercaptoethanol
StainingType: NEGATIVE / Details: Cryo-EM
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Instrument: OTHER / Details: Vitrification instrument: Vitrobot
Method: Blot for 10 seconds before plunging, use 2 layers of filter paper

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Electron microscopy

MicroscopeFEI TECNAI 12
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsCalibrated magnification: 90000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 4.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 90000
Sample stageSpecimen holder: Single tilt cryo holder / Specimen holder model: GATAN LIQUID NITROGEN
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (2k x 2k) / Digitization - Sampling interval: 3.308 µm / Number real images: 99 / Average electron dose: 20 e/Å2 / Details: Data collected on CCD

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Image processing

CTF correctionDetails: Wiener filter on 3D volumes
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 25.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: SPIDER / Number images used: 9183

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Atomic model buiding 1

Initial modelPDB ID:

1ffk

SoftwareName: UCSF Chimera
DetailsRigid-body fit into the density using UCSF Chimera built-in "Fit in Map"
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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