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- EMDB-1212: Hrr25-dependent phosphorylation state regulates organization of t... -

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Basic information

Entry
Database: EMDB / ID: EMD-1212
TitleHrr25-dependent phosphorylation state regulates organization of the pre-40S subunit.
Map datamap of 40S ribosome
Sample
  • Sample: 40S Ribosome
  • Complex: 40S-ribosome
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 32.0 Å
AuthorsSchafer T / Maco B / Petfalski E / Tollervey D / Bottcher B / Aebi U / Hurt E
CitationJournal: Nature / Year: 2006
Title: Hrr25-dependent phosphorylation state regulates organization of the pre-40S subunit.
Authors: Thorsten Schäfer / Bohumil Maco / Elisabeth Petfalski / David Tollervey / Bettina Böttcher / Ueli Aebi / Ed Hurt /
Abstract: The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal ...The formation of eukaryotic ribosomes is a multistep process that takes place successively in the nucleolar, nucleoplasmic and cytoplasmic compartments. Along this pathway, multiple pre-ribosomal particles are generated, which transiently associate with numerous non-ribosomal factors before mature 60S and 40S subunits are formed. However, most mechanistic details of ribosome biogenesis are still unknown. Here we identify a maturation step of the yeast pre-40S subunit that is regulated by the protein kinase Hrr25 and involves ribosomal protein Rps3. A high salt concentration releases Rps3 from isolated pre-40S particles but not from mature 40S subunits. Electron microscopy indicates that pre-40S particles lack a structural landmark present in mature 40S subunits, the 'beak'. The beak is formed by the protrusion of 18S ribosomal RNA helix 33, which is in close vicinity to Rps3. Two protein kinases Hrr25 and Rio2 are associated with pre-40S particles. Hrr25 phosphorylates Rps3 and the 40S synthesis factor Enp1. Phosphorylated Rsp3 and Enp1 readily dissociate from the pre-ribosome, whereas subsequent dephosphorylation induces formation of the beak structure and salt-resistant integration of Rps3 into the 40S subunit. In vivo depletion of Hrr25 inhibits growth and leads to the accumulation of immature 40S subunits that contain unstably bound Rps3. We conclude that the kinase activity of Hrr25 regulates the maturation of 40S ribosomal subunits.
History
DepositionMar 29, 2006-
Header (metadata) releaseApr 3, 2006-
Map releaseJun 1, 2006-
UpdateOct 17, 2012-
Current statusOct 17, 2012Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

1212.gif

Downloads & links

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Map

FileDownload / File: emd_1212.map.gz / Format: CCP4 / Size: 1.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationmap of 40S ribosome
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
5.72 Å/pix.
x 80 pix.
= 457.6 Å		5.72 Å/pix.
x 80 pix.
= 457.6 Å		5.72 Å/pix.
x 80 pix.
= 457.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 5.72 Å
Density

Histogram

Histogram (log scale)
Contour Level1: 0.211 / Movie #1: 0.3
Minimum - Maximum-1.08288 - 2.18498
Average (Standard dev.)0.00655999 (±0.135631)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-40-40
Dimensions808080
Spacing808080
CellA=B=C: 457.6 Å
α=β=γ: 90 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.725.725.72
M x/y/z808080
origin x/y/z0.0000.0000.000
length x/y/z457.600457.600457.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-96-96-96
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS-40-40-40
NC/NR/NS808080
D min/max/mean-1.0832.1850.007

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Supplemental data

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Sample components

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Entire : 40S Ribosome

EntireName: 40S Ribosome
Components
  • Sample: 40S Ribosome
  • Complex: 40S-ribosome

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Supramolecule #1000: 40S Ribosome

SupramoleculeName: 40S Ribosome / type: sample / ID: 1000 / Details: sample was purified from S. cervisiae / Oligomeric state: monomer / Number unique components: 1

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Supramolecule #1: 40S-ribosome

SupramoleculeName: 40S-ribosome / type: complex / ID: 1 / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: SSU 40S
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's Yeast

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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: 7.5 / Details: 50mM Tris-HCl, 100mM NaCl, 10mM MgCl2, 0.5mM DTT
GridDetails: 400 mesh copper rhodium grids (Maxtaform)
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 295 K / Instrument: HOMEMADE PLUNGER
Details: Vitrification instrument: Plunger with environmental chamber
Method: Blot for 15 s with Whatman No 1

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Electron microscopy

MicroscopeFEI/PHILIPS CM200FEG
TemperatureAverage: 94 K
Alignment procedureLegacy - Astigmatism: bjective lens astigmatism was corrected at
Image recordingCategory: CCD / Film or detector model: GENERIC CCD / Digitization - Sampling interval: 14 µm / Number real images: 69 / Bits/pixel: 12
Tilt angle min0
Tilt angle max0
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.2 µm / Nominal magnification: 50000
Sample stageSpecimen holder: Side entry liquid nitrogen-cooled cryo specimen holder
Specimen holder model: GATAN LIQUID NITROGEN

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Image processing

Details236 out of 400 classes were used for final reconstruction
CTF correctionDetails: Each Particle
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMAGIC 5
Details: reconstructions were calculated from half of the class averages. Euler angles of class averages were determined by projection matching
Number images used: 4827
Final angle assignmentDetails: IMAGIC
Final two d classificationNumber classes: 236

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