+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-3151 | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM Structure of the 60S-Arx1-Alb1-Rei1 Complex | |||||||||
Map data | Cryo-EM structure of the 60S-Arx1-Alb1-Rei1 complex (Rei1 C-terminal His6-tag) | |||||||||
Sample |
| |||||||||
Keywords | eukaryotic 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 / hexon binding / 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 / GTP hydrolysis and joining of the 60S ribosomal subunit ...ribosome biogenesis => GO:0042254 / budding cell bud growth / Hydrolases / nucleocytoplasmic transport / hexon binding / 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 / GTP hydrolysis and joining of the 60S ribosomal subunit / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / negative regulation of mRNA splicing, via spliceosome / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / translational elongation / ribosomal large subunit export from nucleus / 90S preribosome / regulation of translational fidelity / protein-RNA complex assembly / translational termination / maturation of LSU-rRNA / Neutrophil degranulation / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ribosomal large subunit biogenesis / translational initiation / macroautophagy / maintenance of translational fidelity / modification-dependent protein catabolic process / protein tag activity / rRNA processing / metallopeptidase activity / ribosome biogenesis / viral capsid / mitotic cell cycle / 5S rRNA binding / large ribosomal subunit rRNA binding / ribosomal large subunit assembly / cytoplasmic translation / cytosolic large ribosomal subunit / sequence-specific DNA binding / negative regulation of translation / rRNA binding / ribosome / protein ubiquitination / structural constituent of ribosome / translation / response to antibiotic / mRNA binding / ubiquitin protein ligase binding / host cell nucleus / nucleolus / proteolysis / RNA binding / zinc ion binding / nucleoplasm / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Saccharomyces cerevisiae (brewer's yeast) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.4 Å | |||||||||
Authors | Greber BJ / Gerhardy S / Leitner A / Leibundgut M / Salem M / Boehringer D / Leulliot N / Aebersold R / Panse VG / Ban N | |||||||||
Citation | Journal: 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 |
|
-Structure visualization
Movie |
Movie viewer |
---|---|
Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_3151.map.gz | 10.2 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-3151-v30.xml emd-3151.xml | 16.2 KB 16.2 KB | Display Display | EMDB header |
Images | EMD_3151_500px.jpg | 182.6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-3151 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-3151 | HTTPS FTP |
-Validation report
Summary document | emd_3151_validation.pdf.gz | 279.2 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_3151_full_validation.pdf.gz | 278.3 KB | Display | |
Data in XML | emd_3151_validation.xml.gz | 5.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3151 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-3151 | HTTPS FTP |
-Related structure data
Related structure data | 5apoMC 3152C 3153C 5apnC M: atomic model generated by this map C: citing same article (ref.) |
---|---|
Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|---|
Related items in Molecule of the Month |
-Map
File | Download / File: emd_3151.map.gz / Format: CCP4 / Size: 37.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Cryo-EM structure of the 60S-Arx1-Alb1-Rei1 complex (Rei1 C-terminal His6-tag) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.39 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
|
-Supplemental data
-Sample components
-Entire : 60S-Arx1-Alb1-Rei1 complex with C-terminal His6-tag on Rei1
Entire | Name: 60S-Arx1-Alb1-Rei1 complex with C-terminal His6-tag on Rei1 |
---|---|
Components |
|
-Supramolecule #1000: 60S-Arx1-Alb1-Rei1 complex with C-terminal His6-tag on Rei1
Supramolecule | Name: 60S-Arx1-Alb1-Rei1 complex with C-terminal His6-tag on Rei1 type: sample / ID: 1000 / Oligomeric state: Stoichiometric 1:1:1:1 assembly / Number unique components: 4 |
---|---|
Molecular weight | Theoretical: 2.4 MDa |
-Supramolecule #1: 60S ribosomal subunit
Supramolecule | Name: 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 GO | 0: GO:0022625 |
Source (natural) | Organism: Saccharomyces cerevisiae (brewer's yeast) / synonym: Baker's yeast / Organelle: Cytosol / Location in cell: Cytosol |
Molecular weight | Theoretical: 2.2 MDa |
-Macromolecule #1: Arx1
Macromolecule | Name: 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 weight | Theoretical: 65 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta 2 DE3 / Recombinant plasmid: pET-15b |
Sequence | UniProtKB: Probable metalloprotease ARX1 / GO: ribosome biogenesis => GO:0042254 |
-Macromolecule #2: Alb1
Macromolecule | Name: 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 weight | Theoretical: 63 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta 2 DE3 / Recombinant plasmid: petM41 |
Sequence | UniProtKB: Ribosome biogenesis protein ALB1 / GO: ribosome biogenesis => GO:0042254 / InterPro: Ribosome biogenesis protein Alb1 |
-Macromolecule #3: Rei1
Macromolecule | Name: Rei1 / type: protein_or_peptide / ID: 3 / 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 / Tissue: Cytosol / Location in cell: Cytosol |
Molecular weight | Theoretical: 45 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) / Recombinant strain: Rosetta DE3 / Recombinant plasmid: pET24a |
Sequence | UniProtKB: Cytoplasmic 60S subunit biogenesis factor REI1 / GO: ribosome biogenesis => GO:0042254 |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.2 mg/mL |
---|---|
Buffer | pH: 8 Details: 20 mM HEPES-KOH pH 8, 100 mM NaCl, 5 mM MgCl2, 5 mM beta-mercaptoethanol |
Grid | Details: Quantifoil holey carbon, glow discharged |
Vitrification | Cryogen name: ETHANE-PROPANE MIXTURE / Chamber temperature: 80 K / Instrument: HOMEMADE PLUNGER |
-Electron microscopy #1
Microscopy ID | 1 |
---|---|
Microscope | FEI TITAN KRIOS |
Details | Data collected using 3-4 exposures per ice hole (2 x 2 or triangular pattern). Movie mode readout in FEI EPU: 7 frames per exosure. |
Date | Mar 20, 2015 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 1440 / Average electron dose: 20 e/Å2 Details: movie mode readout in FEI EPU: 7 frames per exposure |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 100720 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Electron microscopy #2
Microscopy ID | 2 |
---|---|
Microscope | FEI TITAN KRIOS |
Details | Data collected using 3-4 exposures per ice hole (2 x 2 or triangular pattern). Movie mode readout in FEI EPU: 7 frames per exosure. |
Date | Apr 7, 2015 |
Image recording | Category: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Number real images: 2214 / Average electron dose: 20 e/Å2 Details: movie mode readout in FEI EPU: 7 frames per exposure |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Calibrated magnification: 100720 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 0.003 µm / Nominal defocus min: 0.0008 µm / Nominal magnification: 59000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Details | Particles were selected semi-automatically using BATCHBOXER (EMAN). CTF correction using CTFFIND3. Reconstruction using RELION. |
---|---|
CTF correction | Details: per micrograph |
Final reconstruction | Applied symmetry - Point group: C1 (asymmetric) / Algorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: OTHER / Software - Name: CTFFIND3, RELION1.3 / Details: Particles selected in BATCHBOXER (EMAN1.9). / Number images used: 134701 |
-Atomic model buiding 1
Initial model | PDB ID: |
---|---|
Software | Name: UCSF CHIMERA |
Details | The 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). |
Refinement | Space: REAL / Protocol: RIGID BODY FIT |
Output model | PDB-5apo: |