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- EMDB-3153: Cryo-EM structure of the 60S-Arx1-Rei1 complex at near-atomic res... -

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Basic information

Entry
Database: EMDB / ID: EMD-3153
TitleCryo-EM structure of the 60S-Arx1-Rei1 complex at near-atomic resolution
Map dataCryo-EM reconstruction of the 60S-Arx1-Rei1 complex at near-atomic resolution
Sample
  • Sample: 60S-Arx1-Rei1 complex
  • Complex: 60S ribosomal subunit
  • Protein or peptide: Arx1
  • Protein or peptide: Rei1
Keywordseukaryotic ribosome / 60S subunit / ribosome biogenesis / Rei1 / Arx1 / cytoplasmic maturation
Function / homology
Function and homology information


ribosome biogenesis => GO:0042254 / budding cell bud growth / Hydrolases / nucleocytoplasmic transport / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / ribosomal large subunit biogenesis / Neutrophil degranulation / metallopeptidase activity / mitotic cell cycle ...ribosome biogenesis => GO:0042254 / budding cell bud growth / Hydrolases / nucleocytoplasmic transport / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / ribosomal large subunit biogenesis / Neutrophil degranulation / metallopeptidase activity / mitotic cell cycle / sequence-specific DNA binding / nucleolus / proteolysis / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytoplasm
Similarity search - Function
Zinc finger protein 622/Rei1/Reh1 / ZN622/Rei1/Reh1, zinc finger C2H2-type / C2H2 type zinc-finger (2 copies) / : / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Creatinase/aminopeptidase-like / zinc finger / Zinc finger C2H2 superfamily / Zinc finger C2H2 type domain signature. ...Zinc finger protein 622/Rei1/Reh1 / ZN622/Rei1/Reh1, zinc finger C2H2-type / C2H2 type zinc-finger (2 copies) / : / Matrin/U1-C-like, C2H2-type zinc finger / U1-like zinc finger / Creatinase/aminopeptidase-like / zinc finger / Zinc finger C2H2 superfamily / Zinc finger C2H2 type domain signature. / Zinc finger C2H2-type / Winged helix-like DNA-binding domain superfamily
Similarity search - Domain/homology
Cytoplasmic 60S subunit biogenesis factor REI1 / Probable metalloprotease ARX1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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: 0.1
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.1
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD_3153_500...

Downloads & links

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Map

FileDownload / File: emd_3153.map.gz / Format: CCP4 / Size: 37.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM reconstruction of the 60S-Arx1-Rei1 complex at near-atomic resolution
Voxel sizeX=Y=Z: 1.39 Å
Density
Contour LevelBy AUTHOR: 0.1 / Movie #1: 0.1
Minimum - Maximum-0.51979357 - 0.87405241
Average (Standard dev.)0.00580786 (±0.05011464)
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-0.5200.8740.006

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Supplemental data

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Sample components

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Entire : 60S-Arx1-Rei1 complex

EntireName: 60S-Arx1-Rei1 complex
Components
  • Sample: 60S-Arx1-Rei1 complex
  • Complex: 60S ribosomal subunit
  • Protein or peptide: Arx1
  • Protein or peptide: Rei1

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Supramolecule #1000: 60S-Arx1-Rei1 complex

SupramoleculeName: 60S-Arx1-Rei1 complex / type: sample / ID: 1000
Oligomeric state: 1:1:1 stoichiometry of 60S subunit and associated factors
Number unique components: 3
Molecular weightTheoretical: 2.3 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 / Number of copies: 1 / Oligomeric state: Monomer / Recombinant expression: Yes
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: BY4741 / synonym: Baker's yeast / Organelle: Cytosol and nucleus / Location in cell: Cytosol and nucleus
Molecular weightTheoretical: 65 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria) / Recombinant strain: Codon Plus / Recombinant plasmid: pET-47
SequenceUniProtKB: Probable metalloprotease ARX1 / GO: ribosome biogenesis => GO:0042254

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Macromolecule #2: Rei1

MacromoleculeName: Rei1 / type: protein_or_peptide / ID: 2 / Details: C-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: pET-24a
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 R2/1, glow discharged
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber temperature: 80 K / Instrument: HOMEMADE PLUNGER / Method: Manual blotting from the back before plunging.

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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: 2.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
DetailsData collected using 4 exposures per ice hole (2 x 2 pattern). Movie mode readout in FEI EPU: 7 frames per exosure
DateJun 13, 2014
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 1534 / 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: 3.7 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION1.3 / Details: Particles selected in BATCHBOXER (EMAN1.9). / Number images used: 54775
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

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