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- EMDB-3207: Negative stain structure of the Rix1-Ipi1dN50-Ipi3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-3207
TitleNegative stain structure of the Rix1-Ipi1dN50-Ipi3 complex
Map dataReconstruction of mutant Rix1-Ipi1dN50-Ipi3 complex
Sample
  • Sample: Rix1-Ipi1dN50-Ipi3 complex
  • Protein or peptide: Rix1
  • Protein or peptide: Ipi3
  • Protein or peptide: Ipi1dN50
Keywordsribosome / ribosome biogenesis / ribosome assembly / pre-60S / 5S RNP / assembly intermediate / negative-stain / Rix1 complex / Rix1 / Ipi1 / Ipi3
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / negative staining / Resolution: 18.0 Å
AuthorsBarrio-Garcia C / Thoms M / Flemming D / Kater L / Berninghausen O / Bassler J / Beckmann R / Hurt E
CitationJournal: Nat Struct Mol Biol / Year: 2016
Title: Architecture of the Rix1-Rea1 checkpoint machinery during pre-60S-ribosome remodeling.
Authors: Clara Barrio-Garcia / Matthias Thoms / Dirk Flemming / Lukas Kater / Otto Berninghausen / Jochen Baßler / Roland Beckmann / Ed Hurt /
Abstract: Ribosome synthesis is catalyzed by ∼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae ...Ribosome synthesis is catalyzed by ∼200 assembly factors, which facilitate efficient production of mature ribosomes. Here, we determined the cryo-EM structure of a Saccharomyces cerevisiae nucleoplasmic pre-60S particle containing the dynein-related 550-kDa Rea1 AAA(+) ATPase and the Rix1 subcomplex. This particle differs from its preceding state, the early Arx1 particle, by two massive structural rearrangements: an ∼180° rotation of the 5S ribonucleoprotein complex and the central protuberance (CP) rRNA helices, and the removal of the 'foot' structure from the 3' end of the 5.8S rRNA. Progression from the Arx1 to the Rix1 particle was blocked by mutational perturbation of the Rix1-Rea1 interaction but not by a dominant-lethal Rea1 AAA(+) ATPase-ring mutant. After remodeling, the Rix1 subcomplex and Rea1 become suitably positioned to sense correct structural maturation of the CP, which allows unidirectional progression toward mature ribosomes.
History
DepositionOct 22, 2015-
Header (metadata) releaseNov 4, 2015-
Map releaseDec 16, 2015-
UpdateFeb 17, 2016-
Current statusFeb 17, 2016Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.08
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3207_Rix...

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Map

FileDownload / File: emd_3207.map.gz / Format: CCP4 / Size: 21.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of mutant Rix1-Ipi1dN50-Ipi3 complex
Voxel sizeX=Y=Z: 1.65 Å
Density
Contour LevelBy EMDB: 0.084 / Movie #1: 0.08
Minimum - Maximum-0.13170478 - 0.20288256
Average (Standard dev.)0.00068484 (±0.01617203)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 297.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.651.651.65
M x/y/z180180180
origin x/y/z0.0000.0000.000
length x/y/z297.000297.000297.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-180-180-179
NX/NY/NZ360360360
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS180180180
D min/max/mean-0.1320.2030.001

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Supplemental data

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Sample components

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Entire : Rix1-Ipi1dN50-Ipi3 complex

EntireName: Rix1-Ipi1dN50-Ipi3 complex
Components
  • Sample: Rix1-Ipi1dN50-Ipi3 complex
  • Protein or peptide: Rix1
  • Protein or peptide: Ipi3
  • Protein or peptide: Ipi1dN50

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Supramolecule #1000: Rix1-Ipi1dN50-Ipi3 complex

SupramoleculeName: Rix1-Ipi1dN50-Ipi3 complex / type: sample / ID: 1000
Oligomeric state: 2 copies of Rix1, 1 copy of Ipi1 and 2 copies of Ipi3
Number unique components: 3
Molecular weightExperimental: 330 KDa / Theoretical: 334 KDa
Method: size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS)

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Macromolecule #1: Rix1

MacromoleculeName: Rix1 / type: protein_or_peptide / ID: 1 / Name.synonym: Rix1 complex / Number of copies: 2 / Oligomeric state: heterotrimer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast
Molecular weightTheoretical: 867.5 KDa

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Macromolecule #2: Ipi3

MacromoleculeName: Ipi3 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Oligomeric state: heterotrimer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast
Molecular weightExperimental: 61.78 KDa

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Macromolecule #3: Ipi1dN50

MacromoleculeName: Ipi1dN50 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Oligomeric state: heterotrimer / Recombinant expression: No
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: baker's yeast
Molecular weightTheoretical: 37.87 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: 20 mM Hepes, 200 mM NaCl, 10 mM KCl, 10 mM MgCl2 and 1 mM DTT
StainingType: NEGATIVE / Details: stained with 2% (w/v) uranyl acetate
VitrificationCryogen name: NONE / Instrument: OTHER

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Electron microscopy

MicroscopeFEI TECNAI F20
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 1.2 mm / Nominal magnification: 62000
Sample stageSpecimen holder model: SIDE ENTRY, EUCENTRIC
DateMar 6, 2013
Image recordingCategory: CCD / Film or detector model: FEI EAGLE (4k x 4k) / Number real images: 25
Experimental equipment
Model: Tecnai F20 / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 18.0 Å / Resolution method: OTHER / Software - Name: IMAGIC-4D, BOXER / Number images used: 4267
DetailsThe particles were selected using the interactive selection program BOXER

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