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- PDB-6ylf: Rix1-Rea1 pre-60S particle - Rea1, body 3 (rigid body refinement,... -

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Basic information

Entry
Database: PDB / ID: 6ylf
TitleRix1-Rea1 pre-60S particle - Rea1, body 3 (rigid body refinement, composite structure of Rea1 ring and tail)
Components
  • MidasinMDN1
  • Ribosome assembly protein 4
KeywordsRIBOSOME / pre-60S / Biogenesis / LSU / Large subunit / ribosome assembly
Function / homology
Function and homology information


protein-RNA complex remodeling / regulation of ribosomal subunit export from nucleus / Antigen processing: Ubiquitination & Proteasome degradation / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / rRNA processing / ribosomal large subunit assembly / nucleolus / ATP hydrolysis activity / mitochondrion ...protein-RNA complex remodeling / regulation of ribosomal subunit export from nucleus / Antigen processing: Ubiquitination & Proteasome degradation / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / rRNA processing / ribosomal large subunit assembly / nucleolus / ATP hydrolysis activity / mitochondrion / nucleoplasm / ATP binding / nucleus
Similarity search - Function
Midasin / Midasin, AAA lid domain 7 / Midasin AAA lid domain 5 / : / Midasin AAA lid domain / Midasin AAA lid domain / Midasin AAA+ ATPase lid domain / ATPase, dynein-related, AAA domain / AAA domain (dynein-related subfamily) / Sigma-54 interaction domain, ATP-binding site 1 ...Midasin / Midasin, AAA lid domain 7 / Midasin AAA lid domain 5 / : / Midasin AAA lid domain / Midasin AAA lid domain / Midasin AAA+ ATPase lid domain / ATPase, dynein-related, AAA domain / AAA domain (dynein-related subfamily) / Sigma-54 interaction domain, ATP-binding site 1 / NLE / NLE (NUC135) domain / VWFA domain profile. / von Willebrand factor, type A / von Willebrand factor A-like domain superfamily / G-protein beta WD-40 repeat / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Ribosome assembly protein 4 / Midasin
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.2 Å
AuthorsKater, L. / Beckmann, R.
CitationJournal: Mol Cell / Year: 2020
Title: Construction of the Central Protuberance and L1 Stalk during 60S Subunit Biogenesis.
Authors: Lukas Kater / Valentin Mitterer / Matthias Thoms / Jingdong Cheng / Otto Berninghausen / Roland Beckmann / Ed Hurt /
Abstract: Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering ...Ribosome assembly is driven by numerous assembly factors, including the Rix1 complex and the AAA ATPase Rea1. These two assembly factors catalyze 60S maturation at two distinct states, triggering poorly understood large-scale structural transitions that we analyzed by cryo-electron microscopy. Two nuclear pre-60S intermediates were discovered that represent previously unknown states after Rea1-mediated removal of the Ytm1-Erb1 complex and reveal how the L1 stalk develops from a pre-mature nucleolar to a mature-like nucleoplasmic state. A later pre-60S intermediate shows how the central protuberance arises, assisted by the nearby Rix1-Rea1 machinery, which was solved in its pre-ribosomal context to molecular resolution. This revealed a Rix1-Ipi3 tetramer anchored to the pre-60S via Ipi1, strategically positioned to monitor this decisive remodeling. These results are consistent with a general underlying principle that temporarily stabilized immature RNA domains are successively remodeled by assembly factors, thereby ensuring failsafe assembly progression.
History
DepositionApr 7, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 29, 2020Provider: repository / Type: Initial release
Revision 1.1Sep 2, 2020Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Movie
  • Deposited structure unit
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  • EMDB-10837
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Assembly

Deposited unit
AP1: Midasin
xP1: Ribosome assembly protein 4


Theoretical massNumber of molelcules
Total (without water)617,0582
Polymers617,0582
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: immunoprecipitation
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Midasin / MDN1 / Dynein-related AAA-ATPase REA1 / MIDAS-containing protein / Ribosome export/assembly protein 1


Mass: 559951.438 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q12019
#2: Protein Ribosome assembly protein 4 / Notchless protein homolog 1 / Ribosome biogenesis factor RSA4


Mass: 57106.781 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P25382

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Rix1-Rea1 pre-60S assembly particle - Rea1 with the Rsa4-UBL domain
Type: RIBOSOME
Details: Rix1-TAP Flag-Rea1 derived pre-60S assembly complex
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R3/3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 75 e/Å2 / Detector mode: INTEGRATING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 1
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 48 / Used frames/image: 1-48

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Processing

EM software
IDNameVersionCategory
1RELION3.0.8particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
7UCSF Chimeramodel fitting
9RELION3.0.8initial Euler assignment
10RELION3.0.8final Euler assignment
11RELION3.0.8classification
12RELION3.0.83D reconstruction
13PHENIXmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 273799
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 55397 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-ID
16HYP

6hyp

1
26HYD

6hyd

1
36OR5

6or5

1
46QTA

6qta

1

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