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Yorodumi- PDB-7uqb: Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rot... -
+Open data
-Basic information
Entry | Database: PDB / ID: 7uqb | ||||||||||||
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Title | Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a SPB1-D52A strain with AlF4 | ||||||||||||
Components |
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Keywords | RIBOSOME / ribosome biogenesis / K-loop GTPase / GTPase / GTPase transition state / GDP aluminum fluoride | ||||||||||||
Function / homology | Function and homology information regulation of ribosomal subunit export from nucleus / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nuclear exosome (RNase complex) / Hydrolases / PeBoW complex / rRNA primary transcript binding / positive regulation of ATP-dependent activity / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / 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) ...regulation of ribosomal subunit export from nucleus / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / nuclear exosome (RNase complex) / Hydrolases / PeBoW complex / rRNA primary transcript binding / positive regulation of ATP-dependent activity / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / 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) / proteasome binding / maturation of 5.8S rRNA / Major pathway of rRNA processing in the nucleolus and cytosol / SRP-dependent cotranslational protein targeting to membrane / GTP hydrolysis and joining of the 60S ribosomal subunit / ribosomal large subunit binding / 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 / nuclear-transcribed mRNA catabolic process / ATPase activator activity / L13a-mediated translational silencing of Ceruloplasmin expression / preribosome, large subunit precursor / translational elongation / ribosomal large subunit export from nucleus / protein-RNA complex assembly / regulation of translational fidelity / ribosomal subunit export from nucleus / ribonucleoprotein complex binding / endonucleolytic cleavage in ITS1 to separate SSU-rRNA from 5.8S rRNA and LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / Neutrophil degranulation / translation initiation factor activity / maturation of LSU-rRNA / proteasome complex / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / assembly of large subunit precursor of preribosome / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / cytosolic ribosome assembly / ribosomal large subunit biogenesis / ribosomal large subunit assembly / maturation of SSU-rRNA / small-subunit processome / macroautophagy / maintenance of translational fidelity / protein catabolic process / rRNA processing / metallopeptidase activity / protein transport / ribosome biogenesis / protein-macromolecule adaptor activity / ATPase binding / 5S rRNA binding / large ribosomal subunit rRNA binding / cytosolic large ribosomal subunit / cytoplasmic translation / nucleic acid binding / rRNA binding / negative regulation of translation / ribosome / structural constituent of ribosome / translation / GTPase activity / mRNA binding / nucleolus / GTP binding / proteolysis / RNA binding / zinc ion binding / nucleoplasm / identical protein binding / nucleus / metal ion binding / cytosol / cytoplasm Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae BY4741 (yeast) | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.43 Å | ||||||||||||
Authors | Sekulski, K. / Cruz, V.E. / Weirich, C.S. / Erzberger, J.P. | ||||||||||||
Funding support | United States, 3items
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Citation | Journal: Nat Commun / Year: 2023 Title: rRNA methylation by Spb1 regulates the GTPase activity of Nog2 during 60S ribosomal subunit assembly. Authors: Kamil Sekulski / Victor Emmanuel Cruz / Christine S Weirich / Jan P Erzberger / Abstract: Biogenesis of the large ribosomal (60S) subunit involves the assembly of three rRNAs and 46 proteins, a process requiring approximately 70 ribosome biogenesis factors (RBFs) that bind and release the ...Biogenesis of the large ribosomal (60S) subunit involves the assembly of three rRNAs and 46 proteins, a process requiring approximately 70 ribosome biogenesis factors (RBFs) that bind and release the pre-60S at specific steps along the assembly pathway. The methyltransferase Spb1 and the K-loop GTPase Nog2 are essential RBFs that engage the rRNA A-loop during sequential steps in 60S maturation. Spb1 methylates the A-loop nucleotide G2922 and a catalytically deficient mutant strain (spb1) has a severe 60S biogenesis defect. However, the assembly function of this modification is currently unknown. Here, we present cryo-EM reconstructions that reveal that unmethylated G2922 leads to the premature activation of Nog2 GTPase activity and capture a Nog2-GDP-AlF transition state structure that implicates the direct involvement of unmodified G2922 in Nog2 GTPase activation. Genetic suppressors and in vivo imaging indicate that premature GTP hydrolysis prevents the efficient binding of Nog2 to early nucleoplasmic 60S intermediates. We propose that G2922 methylation levels regulate Nog2 recruitment to the pre-60S near the nucleolar/nucleoplasmic phase boundary, forming a kinetic checkpoint to regulate 60S production. Our approach and findings provide a template to study the GTPase cycles and regulatory factor interactions of the other K-loop GTPases involved in ribosome assembly. | ||||||||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 7uqb.cif.gz | 3.5 MB | Display | PDBx/mmCIF format |
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PDB format | pdb7uqb.ent.gz | Display | PDB format | |
PDBx/mmJSON format | 7uqb.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/uq/7uqb ftp://data.pdbj.org/pub/pdb/validation_reports/uq/7uqb | HTTPS FTP |
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-Related structure data
Related structure data | 26686MC 7uooC 7uqzC 7uuiC 7v08C C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
+60S ribosomal protein ... , 36 types, 36 molecules A8BCDEFGHJLMNOPQRSTUVXYZacdefg...
-RNA chain , 4 types, 4 molecules 1236
#2: RNA chain | Mass: 1097942.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: GenBank: 2211411569 |
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#3: RNA chain | Mass: 50682.922 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: GenBank: 1767169448 |
#4: RNA chain | Mass: 38951.105 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: GenBank: 1039023754 |
#6: RNA chain | Mass: 23660.916 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 |
-Protein , 12 types, 12 molecules 5IKWnsvwxyz4
#5: Protein | Mass: 14460.057 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: A0A6A5PUZ0 |
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#16: Protein | Mass: 18546.982 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q08004 |
#18: Protein | Mass: 42596.691 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P38779 |
#30: Protein | Mass: 27098.012 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P33201 |
#47: Protein | Mass: 69984.148 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P53261 |
#52: Protein | Mass: 9226.009 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P40010 |
#55: Protein | Mass: 39665.789 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: A0A6A5PVK5 |
#56: Protein | Mass: 22817.174 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: A0A6A5Q4U5 |
#57: Protein | Mass: 56975.586 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: A0A6A5Q1D2 |
#58: Protein | Mass: 26476.605 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q12522 |
#59: Protein | Mass: 12435.429 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P38202 |
#60: Protein | Mass: 65290.336 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q03862, Hydrolases |
-Ribosome biogenesis protein ... , 6 types, 6 molecules 9oqrtu
#8: Protein | Mass: 19322.037 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: A6ZQK9 |
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#48: Protein | Mass: 25499.186 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P53927 |
#50: Protein | Mass: 52667.832 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q12080 |
#51: Protein | Mass: 29786.783 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P40078 |
#53: Protein | Mass: 36621.074 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P40693 |
#54: Protein | Mass: 24027.650 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q07915 |
-Nucleolar GTP-binding protein ... , 2 types, 2 molecules bm
#35: Protein | Mass: 74531.227 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q02892 |
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#46: Protein | Mass: 55585.590 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P53742 |
-Non-polymers , 7 types, 467 molecules
#61: Chemical | #62: Chemical | ChemComp-MG / #63: Chemical | ChemComp-ZN / #64: Chemical | #65: Chemical | ChemComp-ALF / | #66: Chemical | ChemComp-K / | #67: Water | ChemComp-HOH / | |
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-Details
Has ligand of interest | Y |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Nucleolar 60S intermediate purified with tags on Tif6 and Nog2 from a SPB1-D52A strain. Type: RIBOSOME / Entity ID: #1-#34, #36-#60 / Source: NATURAL | ||||||||||||||||||||||||||||||||||||||||
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Molecular weight | Value: 3.3 MDa / Experimental value: NO | ||||||||||||||||||||||||||||||||||||||||
Source (natural) | Organism: Saccharomyces cerevisiae BY4741 (yeast) | ||||||||||||||||||||||||||||||||||||||||
Buffer solution | pH: 8 | ||||||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1 | ||||||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 900 nm / Calibrated defocus min: 900 nm / Calibrated defocus max: 2200 nm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 0.05 sec. / Electron dose: 1.25 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7614 |
-Processing
Software |
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 790530 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.43 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 414994 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Protocol: RIGID BODY FIT | ||||||||||||||||||||||||
Atomic model building | PDB-ID: 3JCT Accession code: 3JCT / Source name: PDB / Type: experimental model | ||||||||||||||||||||||||
Refinement | Cross valid method: NONE Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2 | ||||||||||||||||||||||||
Displacement parameters | Biso mean: 19.92 Å2 | ||||||||||||||||||||||||
Refine LS restraints |
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