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- EMDB-0330: Rea1 Wild type ADP state (AAA+ ring + stem) -

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Basic information

Entry
Database: EMDB / ID: 0330
TitleRea1 Wild type ADP state (AAA+ ring + stem)
Map data
SampleRea1 (MIDASIN) ring with ADP:
SourceSaccharomyces cerevisiae (baker's yeast)
Methodsingle particle reconstruction / cryo EM / 4.4 Å resolution
AuthorsSosnowski P / Urnavicius L / Boland A / Fagiewicz R / Busselez J / Papai G / Schmidt H
CitationJournal: Elife / Year: 2018
Title: The CryoEM structure of the ribosome maturation factor Rea1.
Authors: Piotr Sosnowski / Linas Urnavicius / Andreas Boland / Robert Fagiewicz / Johan Busselez / Gabor Papai / Helgo Schmidt
Abstract: The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly ...The biogenesis of 60S ribosomal subunits is initiated in the nucleus where rRNAs and proteins form pre-60S particles. These pre-60S particles mature by transiently interacting with various assembly factors. The ~5000 amino-acid AAA+ ATPase Rea1 (or Midasin) generates force to mechanically remove assembly factors from pre-60S particles, which promotes their export to the cytosol. Here we present three Rea1 cryoEM structures. We visualise the Rea1 engine, a hexameric ring of AAA+ domains, and identify an α-helical bundle of AAA2 as a major ATPase activity regulator. The α-helical bundle interferes with nucleotide-induced conformational changes that create a docking site for the substrate binding MIDAS domain on the AAA +ring. Furthermore, we reveal the architecture of the Rea1 linker, which is involved in force generation and extends from the AAA+ ring. The data presented here provide insights into the mechanism of one of the most complex ribosome maturation factors.
DateDeposition: Oct 31, 2018 / Header (metadata) release: Dec 5, 2018 / Map release: Dec 5, 2018 / Last update: Dec 5, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0106
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0106
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0330.map.gz (map file in CCP4 format, 226493 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
384 pix
1.1 Å/pix.
= 422.4 Å
384 pix
1.1 Å/pix.
= 422.4 Å
384 pix
1.1 Å/pix.
= 422.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density
Contour Level:0.0106 (by author), 0.0106 (movie #1):
Minimum - Maximum-0.0069332263 - 0.036207095
Average (Standard dev.)0.000097644916 (0.0011070691)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions384384384
Origin0.00.00.0
Limit383.0383.0383.0
Spacing384384384
CellA=B=C: 422.40002 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z422.400422.400422.400
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ450450450
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0070.0360.000

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Supplemental data

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Sample components

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Entire Rea1 (MIDASIN) ring with ADP

EntireName: Rea1 (MIDASIN) ring with ADP / Number of components: 1

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Component #1: protein, Rea1 (MIDASIN) ring with ADP

ProteinName: Rea1 (MIDASIN) ring with ADP / Recombinant expression: No
SourceSpecies: Saccharomyces cerevisiae (baker's yeast)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/ml / Buffer solution: ADP was added 5 minute before the plunging / pH: 7.2
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 46.2 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 105000.0 X (nominal), 105000.0 X (calibrated)
Cs: 0.01 mm / Imaging mode: BRIGHT FIELD / Defocus: 1800.0 - 3400.0 nm / Energy filter: GIF Quantum LS
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 23230 / Sampling size: 5 microns

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 76071
3D reconstructionSoftware: RELION / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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