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- EMDB-3152: Cryo-EM structure of the 60S-Arx1-Alb1-Rei1 complex (Rei1 N-termi... -

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Basic information

Entry
Database: EMDB / ID: EMD-3152
TitleCryo-EM structure of the 60S-Arx1-Alb1-Rei1 complex (Rei1 N-terminal His6-tag)
Map dataCryo-EM structure of the 60S-Arx1-Alb1-Rei1 complex (Rei1 N-terminal His6-tag)
Sample
  • Sample: 60S-Arx1-Alb1-Rei1 complex with N-terminal His6-tag on Rei1
  • Complex: 60S ribosomal subunit
  • Protein or peptide: Arx1
  • Protein or peptide: Alb1
  • Protein or peptide: Rei1
Keywordseukaryotic ribosome / 60S subunit / ribosome biogenesis / Rei1 / Arx1 / Alb1 / cytoplasmic maturation
Function / homology
Function and homology information


ribosome biogenesis => GO:0042254 / budding cell bud growth / Hydrolases / nucleocytoplasmic transport / 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) / response to cycloheximide / SRP-dependent cotranslational protein targeting to membrane / 90S preribosome / GTP hydrolysis and joining of the 60S ribosomal subunit ...ribosome biogenesis => GO:0042254 / budding cell bud growth / Hydrolases / nucleocytoplasmic transport / 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) / response to cycloheximide / SRP-dependent cotranslational protein targeting to membrane / 90S preribosome / GTP hydrolysis and joining of the 60S ribosomal subunit / Formation of a pool of free 40S subunits / 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 / translational termination / maturation of LSU-rRNA / ribosomal large subunit biogenesis / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Neutrophil degranulation / maintenance of translational fidelity / macroautophagy / modification-dependent protein catabolic process / protein tag activity / rRNA processing / ribosomal large subunit assembly / metallopeptidase activity / large ribosomal subunit rRNA binding / ribosome biogenesis / mitotic cell cycle / 5S rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / negative regulation of translation / sequence-specific DNA binding / rRNA binding / protein ubiquitination / ribosome / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / ubiquitin protein ligase binding / nucleolus / proteolysis / RNA binding / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ribosome biogenesis protein Alb1 / Ribosome biogenesis protein Alb1 / Alb1 / Zinc finger protein 622/Rei1/Reh1 / ZN622/Rei1/Reh1, zinc finger C2H2-type / C2H2 type zinc-finger (2 copies) / : / 60s Acidic ribosomal protein / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger ...Ribosome biogenesis protein Alb1 / Ribosome biogenesis protein Alb1 / Alb1 / Zinc finger protein 622/Rei1/Reh1 / ZN622/Rei1/Reh1, zinc finger C2H2-type / C2H2 type zinc-finger (2 copies) / : / 60s Acidic ribosomal protein / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / 60S acidic ribosomal protein P0 / Creatinase/aminopeptidase-like / : / 50S ribosomal protein L10, insertion domain superfamily / 60S ribosomal protein L10P, insertion domain / Insertion domain in 60S ribosomal protein L10P / Ribosomal protein L29e / Ribosomal protein L10e, conserved site / Ribosomal protein L10e / Ribosomal protein L27e, conserved site / Ribosomal L29e protein family / Ribosomal protein L24e, conserved site / Ribosomal protein L34e, conserved site / Eukaryotic Ribosomal Protein L27, KOW domain / Ribosomal protein L44e / Ribosomal protein L38e / Ribosomal protein L38e superfamily / Ribosomal protein L27e / Ribosomal protein L27e superfamily / Ribosomal L38e protein family / Ribosomal protein L23/L25, N-terminal / 60S ribosomal protein L35 / Ribosomal protein L35Ae, conserved site / Ribosomal protein L30e, conserved site / Ribosomal protein L44 / 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 / zinc finger / Ribosomal protein L22e / Ribosomal protein L22e superfamily / Ribosomal L22e protein family / Ribosomal protein L30/YlxQ / Ribosomal protein L34e / Ribosomal protein L31e, conserved site / Ribosomal protein L37ae / 60S ribosomal protein L6E / Ribosomal protein L19, eukaryotic / Ribosomal protein L35A / Ribosomal protein L36e / Ribosomal protein L36e domain superfamily / Ribosomal L40e family / Ribosomal protein L36e / Ribosomal protein L35A superfamily / Ribosomal protein L44e signature. / Ribosomal_L40e / Ribosomal protein L40e / Ribosomal protein L40e superfamily / Ribosomal protein L7A/L8 / Ribosomal protein L27e signature. / Ribosomal protein L10e signature. / Ribosomal protein L13e / Ribosomal protein L13e / 60S ribosomal protein L4, C-terminal domain / 60S ribosomal protein L18a/ L20, eukaryotes / Ribosomal protein L6e / Ribosomal protein L19/L19e conserved site / Ribosomal L37ae protein family / 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 / 60S ribosomal protein L4 C-terminal domain / Ribosomal protein L19e signature. / Ribosomal proteins 50S-L18Ae/60S-L20/60S-L18A / Ribosomal protein L24e signature. / Ribosomal protein L5 eukaryotic, C-terminal / Ribosomal L18 C-terminal region / Ribosomal protein L34e signature. / Ribosomal protein L31e / Ribosomal protein L31e domain superfamily / Ribosomal protein L6e signature. / Ribosomal_L31e / Ribosomal protein L15e, conserved site / Ribosomal protein L30e signature 1. / Ribosomal protein L21e / Ribosomal protein L21e, conserved site / Ribosomal protein L21 superfamily / Ribosomal protein 60S L18 and 50S L18e / Ribosomal protein L30e signature 2. / 60S ribosomal protein L19 / Ribosomal protein L4/L1e, eukaryotic/archaeal, conserved site / Ribosomal protein L37e, conserved site / Ribosomal protein L3, domain 3, archaeal type superfamily / Ribosomal protein L3, archaeal/eukaryotic type / Ribosomal protein L36e signature. / Ribosomal protein L37e
Similarity search - Domain/homology
Large ribosomal subunit protein uL15 / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / 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 uL15 / Large ribosomal subunit protein eL24A / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL39 / Large ribosomal subunit protein uL10 / 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 eL29 / 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 / Ubiquitin-ribosomal protein eL40A fusion protein / Large ribosomal subunit protein eL20A / Large ribosomal subunit protein eL43A / Large ribosomal subunit protein eL42A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL2A / Large ribosomal subunit protein eL18A / 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 / Large ribosomal subunit protein uL18 / Large ribosomal subunit protein uL13A / Large ribosomal subunit protein eL14A / Large ribosomal subunit protein eL32 / Cytoplasmic 60S subunit biogenesis factor REI1 / Large ribosomal subunit protein uL16 / Ribosome biogenesis protein ALB1 / Large ribosomal subunit protein eL37A / Large ribosomal subunit protein eL38 / Large ribosomal subunit protein eL34A / Large ribosomal subunit protein eL6A / Large ribosomal subunit protein eL21A / Probable metalloprotease ARX1 / Large ribosomal subunit protein eL13A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsGreber BJ / Gerhardy S / Leitner A / Leibundgut M / Salem M / Boehringer D / Leulliot N / Aebersold R / Panse VG / Ban N
CitationJournal: Cell / Year: 2016
Title: Insertion of the Biogenesis Factor Rei1 Probes the Ribosomal Tunnel during 60S Maturation.
Authors: Basil Johannes Greber / Stefan Gerhardy / Alexander Leitner / Marc Leibundgut / Michèle Salem / Daniel Boehringer / Nicolas Leulliot / Ruedi Aebersold / Vikram Govind Panse / Nenad Ban /
Abstract: Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, ...Eukaryotic ribosome biogenesis depends on several hundred assembly factors to produce functional 40S and 60S ribosomal subunits. The final phase of 60S subunit biogenesis is cytoplasmic maturation, which includes the proofreading of functional centers of the 60S subunit and the release of several ribosome biogenesis factors. We report the cryo-electron microscopy (cryo-EM) structure of the yeast 60S subunit in complex with the biogenesis factors Rei1, Arx1, and Alb1 at 3.4 Å resolution. In addition to the network of interactions formed by Alb1, the structure reveals a mechanism for ensuring the integrity of the ribosomal polypeptide exit tunnel. Arx1 probes the entire set of inner-ring proteins surrounding the tunnel exit, and the C terminus of Rei1 is deeply inserted into the ribosomal tunnel, where it forms specific contacts along almost its entire length. We provide genetic and biochemical evidence that failure to insert the C terminus of Rei1 precludes subsequent steps of 60S maturation.
History
DepositionSep 15, 2015-
Header (metadata) releaseSep 30, 2015-
Map releaseDec 16, 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: 0.06
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5apn
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD_3152_500...

Downloads & links

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Map

FileDownload / File: emd_3152.map.gz / Format: CCP4 / Size: 37.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of the 60S-Arx1-Alb1-Rei1 complex (Rei1 N-terminal His6-tag)
Voxel sizeX=Y=Z: 1.39 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.14021601 - 0.35198492
Average (Standard dev.)0.00677288 (±0.02809288)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 300.24 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.391.391.39
M x/y/z216216216
origin x/y/z0.0000.0000.000
length x/y/z300.240300.240300.240
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/NS216216216
D min/max/mean-0.1400.3520.007

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Supplemental data

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Sample components

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Entire : 60S-Arx1-Alb1-Rei1 complex with N-terminal His6-tag on Rei1

EntireName: 60S-Arx1-Alb1-Rei1 complex with N-terminal His6-tag on Rei1
Components
  • Sample: 60S-Arx1-Alb1-Rei1 complex with N-terminal His6-tag on Rei1
  • Complex: 60S ribosomal subunit
  • Protein or peptide: Arx1
  • Protein or peptide: Alb1
  • Protein or peptide: Rei1

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Supramolecule #1000: 60S-Arx1-Alb1-Rei1 complex with N-terminal His6-tag on Rei1

SupramoleculeName: 60S-Arx1-Alb1-Rei1 complex with N-terminal His6-tag on Rei1
type: sample / ID: 1000 / Oligomeric state: Stoichiometric 1:1:1:1 assembly / Number unique components: 4
Molecular weightTheoretical: 2.4 MDa

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Supramolecule #1: 60S ribosomal subunit

SupramoleculeName: 60S ribosomal subunit / type: complex / ID: 1 / Recombinant expression: No
Ribosome-details: ribosome-eukaryote: LSU 60S, LSU RNA 28S, LSU RNA 5.8S, LSU RNA 5S
Ref GOdivclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp ...
divclassse qspanoncli ckpopupspa nclassgree n(this)spandata popltspanc lassquotlo adingbarqu otgtltimgs rcquotimgl oadinggifq uotdecodin gquotasync quotgtltsp angtdataur lajaxphp?m odetaxoamp kGO3A00226 25ampajax1 classpoptr giGO002262 5ispandiv
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Organelle: Cytosol / Location in cell: Cytosol
Molecular weightTheoretical: 2.2 MDa

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Macromolecule #1: Arx1

MacromoleculeName: Arx1 / type: protein_or_peptide / ID: 1 / Details: N-terminal His6-tag / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Organelle: Cytosol and nucleus / Location in cell: Cytosol and nucleus
Molecular weightTheoretical: 65 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2 / Recombinant plasmid: pET-15b
SequenceUniProtKB: Probable metalloprotease ARX1 / GO: ribosome biogenesis => GO:0042254

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Macromolecule #2: Alb1

MacromoleculeName: Alb1 / type: protein_or_peptide / ID: 2
Details: N-terminal maltose binding protein tag; co-expressed with Arx1
Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Organelle: Cytosol and nucleus / Location in cell: Cytosol and nucleus
Molecular weightTheoretical: 63 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta 2 / Recombinant plasmid: petM41
SequenceUniProtKB: Ribosome biogenesis protein ALB1 / GO: ribosome biogenesis => GO:0042254 / InterPro: Ribosome biogenesis protein Alb1

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Macromolecule #3: Rei1

MacromoleculeName: Rei1 / type: protein_or_peptide / ID: 3 / Details: N-terminal His6-tag / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Organelle: Cytosol / Location in cell: Cytosol
Molecular weightTheoretical: 45 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: Rosetta / Recombinant plasmid: pProEX-htb
SequenceUniProtKB: Cytoplasmic 60S subunit biogenesis factor REI1 / GO: ribosome biogenesis => GO:0042254

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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

Concentration0.2 mg/mL
BufferpH: 8
Details: 20 mM HEPES-KOH pH 8, 100 mM NaCl, 5 mM MgCl2, 5 mM beta-mercaptoethanol
GridDetails: Quantifoil holey carbon R2/2, glow discharged
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber temperature: 80 K / Instrument: HOMEMADE PLUNGER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 100720 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DetailsData acquired using 4 spots (2 x 2) per ice hole. Movie mode readout in FEI EPU: 7 frames per exosure
DateApr 17, 2015
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 670 / Average electron dose: 20 e/Å2
Details: movie mode readout in FEI EPU: 7 frames per exposure
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: per micrograph
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION1.3 / Details: Particles selected in BATCHBOXER (EMAN1.9). / Number images used: 22040
DetailsParticles were selected semi-automatically using BATCHBOXER (EMAN). CTF correction using CTFFIND3. Reconstruction using RELION.

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Atomic model buiding 1

Initial modelPDB ID:

4v8t

SoftwareName: UCSF Chimera
DetailsThe coordinate model of the lower-resolution structure of the 60S-Arx1-Rei1 complex was fitted into the cryo-EM density using UCSF CHIMERA. The model was then adjusted using COOT (RNA) and O (proteins).
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-5apn:
Structure of the yeast 60S ribosomal subunit in complex with Arx1, Alb1 and N-terminally tagged Rei1

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