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- EMDB-3199: Cryo-EM structure of the Rix1-Rea1 pre-60S particle -

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Basic information

Entry
Database: EMDB / ID: EMD-3199
TitleCryo-EM structure of the Rix1-Rea1 pre-60S particle
Map dataCryo-EM structure of the Rix1-Rea1 pre-60S particle
Sample
  • Sample: Rix1-Rea1 pre60S particle
  • Complex: pre-60S particle
Keywordsribosome / ribosome biogenesis / ribosome assembly / pre-60S / 5S RNP / assembly intermediate / cryo-EM / Rea1 / Rix1 complex / Sda1
Function / homology
Function and homology information


protein-RNA complex remodeling / regulation of ribosomal subunit export from nucleus / Hydrolases / Antigen processing: Ubiquitination & Proteasome degradation / traversing start control point of mitotic cell cycle / positive regulation of ATP-dependent activity / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nuclear-transcribed mRNA catabolic process / pre-mRNA 5'-splice site binding / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) ...protein-RNA complex remodeling / regulation of ribosomal subunit export from nucleus / Hydrolases / Antigen processing: Ubiquitination & Proteasome degradation / traversing start control point of mitotic cell cycle / positive regulation of ATP-dependent activity / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nuclear-transcribed mRNA catabolic process / pre-mRNA 5'-splice site binding / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of 5.8S rRNA / ribosomal subunit export from nucleus / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / ATPase activator activity / Formation of a pool of free 40S subunits / ribosomal large subunit binding / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / negative regulation of mRNA splicing, via spliceosome / protein-RNA complex assembly / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal large subunit export from nucleus / regulation of translational fidelity / maturation of SSU-rRNA / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA / ribosomal large subunit biogenesis / Neutrophil degranulation / translation initiation factor activity / small-subunit processome / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / maintenance of translational fidelity / macroautophagy / ribosomal large subunit assembly / rRNA processing / metallopeptidase activity / large ribosomal subunit rRNA binding / protein transport / ribosome biogenesis / cytoplasmic translation / 5S rRNA binding / cytosolic large ribosomal subunit / ATPase binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / cell cycle / translation / mRNA binding / GTPase activity / GTP binding / nucleolus / ATP hydrolysis activity / mitochondrion / proteolysis / RNA binding / nucleoplasm / ATP binding / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
SDA1 domain / Midasin / Uncharacterised domain NUC130/133, N-terminal / Sda1 / Midasin, AAA lid domain 7 / Midasin AAA lid domain 5 / : / : / SDA1, conserved domain / SDA1, HEAT repeat ...SDA1 domain / Midasin / Uncharacterised domain NUC130/133, N-terminal / Sda1 / Midasin, AAA lid domain 7 / Midasin AAA lid domain 5 / : / : / SDA1, conserved domain / SDA1, HEAT repeat / Midasin AAA lid domain / Midasin AAA lid domain / Midasin AAA+ ATPase lid domain / SDA1, C-terminal / : / ATPase, dynein-related, AAA domain / AAA domain (dynein-related subfamily) / Sigma-54 interaction domain, ATP-binding site 1 / NLE / NLE (NUC135) domain / Ribosome assembly factor Mrt4 / NOG, C-terminal / Nucleolar GTP-binding protein 1 / NOGCT (NUC087) domain / Nucleolar GTP-binding protein 1, Rossman-fold domain / NOG1, N-terminal helical domain / Nucleolar GTP-binding protein 1 (NOG1) / NOG1 N-terminal helical domain / OBG-type guanine nucleotide-binding (G) domain / OBG-type guanine nucleotide-binding (G) domain profile. / Translation initiation factor IF6 / eIF-6 family / translation initiation factor 6 / Creatinase/aminopeptidase-like / GTP binding domain / 50S ribosomal protein L10, insertion domain superfamily / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / metallochaperone-like domain / TRASH domain / Ribosomal protein L27e, conserved site / : / Ribosomal protein L34e, conserved site / Ribosomal protein L1, conserved site / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal protein L27e / Ribosomal protein L27e superfamily / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L38e protein family / Ribosomal L22e protein family / Ribosomal protein L23/L25, N-terminal / 60S ribosomal protein L35 / Ribosomal protein L1 / Ribosomal protein L35Ae, conserved site / Ribosomal protein L30e, conserved site / Ribosomal protein L34Ae / Ribosomal protein L23, N-terminal domain / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal L27e protein family / Ribosomal Protein L6, KOW domain / Ribosomal protein L30/YlxQ / Ribosomal protein L13e / Ribosomal protein L13e / Ribosomal protein L34e / Ribosomal protein L31e, conserved site / Ribosomal protein L1 signature. / Ribosomal protein L37ae / Ribosomal protein L14e domain / 60S ribosomal protein L6E / Ribosomal protein L19, eukaryotic / Ribosomal protein L35A / Ribosomal protein L36e / Ribosomal protein L36e domain superfamily / Ribosomal protein L36e / Ribosomal protein L35A superfamily / VWFA domain profile. / Ribosomal protein L7A/L8 / Ribosomal protein L27e signature. / Ribosomal protein L32e, conserved site / 60S ribosomal protein L4, C-terminal domain / 60S ribosomal protein L18a/ L20, eukaryotes / Ribosomal protein L7, eukaryotic / Ribosomal protein L6e / Ribosomal protein L14 / Ribosomal protein L30, N-terminal / Ribosomal protein L6, conserved site-2 / Ribosomal protein L14, KOW motif / Ribosomal L30 N-terminal domain / Ribosomal protein L19/L19e conserved site / Ribosomal protein L14 / Ribosomal L37ae protein family / Ribosomal protein L1, 3-layer alpha/beta-sandwich / Ribosomal protein L35Ae / 50S ribosomal protein L18Ae/60S ribosomal protein L20 and L18a / Ribosomal protein L39e, conserved site / Ribosomal protein 50S-L18Ae/60S-L20/60S-L18A
Similarity search - Domain/homology
Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL15A ...Large ribosomal subunit protein uL15 / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL30A / Large ribosomal subunit protein uL6A / Large ribosomal subunit protein uL22A / Large ribosomal subunit protein uL24A / Large ribosomal subunit protein eL33A / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL22A / Large ribosomal subunit protein uL5A / Large ribosomal subunit protein eL27A / Large ribosomal subunit protein eL31A / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL1A / Large ribosomal subunit protein uL2A / Large ribosomal subunit protein eL18A / Large ribosomal subunit protein uL11A / Large ribosomal subunit protein eL19A / Large ribosomal subunit protein uL29A / Large ribosomal subunit protein uL4A / Large ribosomal subunit protein eL30 / Large ribosomal subunit protein uL3 / Large ribosomal subunit protein eL8A / Ribosome assembly protein 4 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL13A / Ribosome assembly factor MRT4 / Large ribosomal subunit protein eL14A / Large ribosomal subunit protein eL32 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Protein SDA1 / Large ribosomal subunit protein eL34A / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Nucleolar GTP-binding protein 1 / Probable metalloprotease ARX1 / Ribosome biogenesis protein RLP24 / Midasin / Eukaryotic translation initiation factor 6 / Large ribosomal subunit protein eL13A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 9.5 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 19, 2015-
Header (metadata) releaseNov 4, 2015-
Map releaseDec 9, 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: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5jcs
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5jcs
  • Surface level: 0.3
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3199_Rix...

Downloads & links

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Map

FileDownload / File: emd_3199.map.gz / Format: CCP4 / Size: 238.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the Rix1-Rea1 pre-60S particle
Voxel sizeX=Y=Z: 2.069 Å
Density
Contour LevelBy AUTHOR: 0.3 / Movie #1: 0.3
Minimum - Maximum-0.72063726 - 1.47908759
Average (Standard dev.)0.00007106 (±0.04510443)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 827.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.0692.0692.069
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z827.600827.600827.600
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/NS400400400
D min/max/mean-0.7211.4790.000

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Supplemental data

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Sample components

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Entire : Rix1-Rea1 pre60S particle

EntireName: Rix1-Rea1 pre60S particle
Components
  • Sample: Rix1-Rea1 pre60S particle
  • Complex: pre-60S particle

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Supramolecule #1000: Rix1-Rea1 pre60S particle

SupramoleculeName: Rix1-Rea1 pre60S particle / type: sample / ID: 1000 / Number unique components: 1

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Supramolecule #1: pre-60S particle

SupramoleculeName: pre-60S particle / type: complex / ID: 1 / Name.synonym: pre-60S / Recombinant expression: No / Ribosome-details: ribosome-eukaryote: LSU 60S
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: 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

Concentration2.06 mg/mL
BufferpH: 7.5 / Details: 50 mM Tris-HCl, 100 mM NaCl, 1.5 mM or 5 mM MgCl2
GridDetails: The freshly prepared pre-60S sample was applied to 2 nm pre-coated Quantifoil holey carbon supported grids and vitrified
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Instrument: FEI VITROBOT MARK IV / Method: Blot for 3 seconds before plunging

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 1.1 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DateJun 12, 2014
Image recordingCategory: CCD / Film or detector model: TVIPS TEMCAM-F816 (8k x 8k) / Number real images: 2659 / Average electron dose: 20 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Subvolumes
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 9.5 Å / Resolution method: OTHER / Software - Name: Signature, Spider / Number images used: 15749
DetailsThe particles were selected using the automatic selection program SIGNATURE

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Atomic model buiding 1

Initial modelPDB ID:

3j64
PDB Unreleased entry

SoftwareName: Chimera, MDFF, Coot
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5jcs:
CRYO-EM STRUCTURE OF THE RIX1-REA1 PRE-60S PARTICLE

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Atomic model buiding 2

Initial modelPDB ID:

3u5d
PDB Unreleased entry

SoftwareName: Chimera, MDFF, Coot
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5jcs:
CRYO-EM STRUCTURE OF THE RIX1-REA1 PRE-60S PARTICLE

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Atomic model buiding 3

Initial modelPDB ID:

3u5e
PDB Unreleased entry

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5jcs:
CRYO-EM STRUCTURE OF THE RIX1-REA1 PRE-60S PARTICLE

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Atomic model buiding 4

Initial modelPDB ID:

3j65
PDB Unreleased entry

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5jcs:
CRYO-EM STRUCTURE OF THE RIX1-REA1 PRE-60S PARTICLE

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Atomic model buiding 5

Initial modelPDB ID:

4wjs

SoftwareName: MDFF, Coot
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-5jcs:
CRYO-EM STRUCTURE OF THE RIX1-REA1 PRE-60S PARTICLE

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