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- PDB-6hyd: Rea1 Wild type ADP state (tail part) -

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Entry
Database: PDB / ID: 6hyd
TitleRea1 Wild type ADP state (tail part)
ComponentsMidasin,Midasin,Midasin
KeywordsMOTOR PROTEIN / Rea1 / Mdn1 / Midasin / AAA+ protein / ribosome maturation / molecular machine
Function / homologyvon Willebrand factor, type A / ATPase, dynein-related, AAA domain / VWFA domain profile. / AAA domain (dynein-related subfamily) / von Willebrand factor A-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Sigma-54 interaction domain, ATP-binding site 1 / Midasin / AAA+ ATPase domain / regulation of ribosomal subunit export from nucleus ...von Willebrand factor, type A / ATPase, dynein-related, AAA domain / VWFA domain profile. / AAA domain (dynein-related subfamily) / von Willebrand factor A-like domain superfamily / P-loop containing nucleoside triphosphate hydrolase / Sigma-54 interaction domain, ATP-binding site 1 / Midasin / AAA+ ATPase domain / regulation of ribosomal subunit export from nucleus / protein-containing complex remodeling / rRNA processing / ribosomal large subunit assembly / ATPase activity / nucleolus / mitochondrion / nucleoplasm / ATP binding / nucleus / Midasin
Function and homology information
Specimen sourceSaccharomyces cerevisiae (baker's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.9 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 19, 2018 / Release: Dec 12, 2018

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Structure visualization

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Assembly

Deposited unit
A: Midasin,Midasin,Midasin


Theoretical massNumber of molelcules
Total (without water)192,0461
Polyers192,0461
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)0
ΔGint (kcal/M)0
Surface area (Å2)80610
MethodPISA

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Components

#1: Protein/peptide Midasin,Midasin,Midasin / Dynein-related AAA-ATPase REA1 / MIDAS-containing protein / Ribosome export/assembly protein 1


Mass: 192045.828 Da / Num. of mol.: 1
Source: (gene. exp.) Saccharomyces cerevisiae (baker's yeast)
Strain: ATCC 204508 / S288c / Gene: MDN1, REA1, YLR106C, L2901, L8004.13 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: Q12019

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Rea1 (MIDASIN) tail with ADP / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (baker's yeast)
Buffer solutionDetails: ADP was added 5 minute before the plunging / pH: 7.2
Buffer component
IDConc.NameFormulaBuffer ID
1150 mMHEPESC8H18N2O4S1
210 mMMagnesium AcetateMg(CH3COO)21
35 mMEGTAC14H24N2O101
45 mMDTTC4H10O2S21
53 mMADPC10H15N5O10P21
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 / Grid type: Quantifoil R2/2
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 105000 / Calibrated magnification: 105000 / Nominal defocus max: 3400 nm / Nominal defocus min: 1800 nm / Cs: 0.01 mm / Alignment procedure: ZEMLIN TABLEAU
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Residual tilt: 12 mradians
Image recordingAverage exposure time: 0.2 sec. / Electron dose: 46.2 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 14 / Number of real images: 23230
EM imaging opticsEnergyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV / Sph aberration corrector: Titan Krios Cs Corrector
Image scansSampling size: 5 microns / Width: 3712 / Height: 3840 / Movie frames/image: 35 / Used frames/image: 2-35

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
5RELIONCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 432556 / Symmetry type: POINT
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412396
ELECTRON MICROSCOPYf_angle_d0.80616802
ELECTRON MICROSCOPYf_dihedral_angle_d5.1617427
ELECTRON MICROSCOPYf_chiral_restr0.0471989
ELECTRON MICROSCOPYf_plane_restr0.0062084

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