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- EMDB-26941: Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rot... -
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Basic information
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Title | Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a Spb1 D52A suppressor 3 strain | ||||||||||||
![]() | Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a Spb1 D52A suppressor 3 strain | ||||||||||||
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![]() | ribosome biogenesis / K-loop GTPase / GTPase / RIBOSOME | ||||||||||||
Function / homology | ![]() regulation of ribosomal subunit export from nucleus / nuclear exosome (RNase complex) / Hydrolases / PeBoW complex / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / pre-mRNA 5'-splice site binding / maturation of 5.8S rRNA / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / proteasome binding / Major pathway of rRNA processing in the nucleolus and cytosol ...regulation of ribosomal subunit export from nucleus / nuclear exosome (RNase complex) / Hydrolases / PeBoW complex / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / pre-mRNA 5'-splice site binding / maturation of 5.8S rRNA / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / proteasome binding / 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 / 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 / Formation of a pool of free 40S subunits / preribosome, large subunit precursor / nuclear-transcribed mRNA catabolic process / L13a-mediated translational silencing of Ceruloplasmin expression / translational elongation / ribosomal large subunit export from nucleus / ribosomal subunit export from nucleus / regulation of translational fidelity / 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) / protein-RNA complex assembly / maturation of LSU-rRNA / translation initiation factor activity / Neutrophil degranulation / proteasome complex / assembly of large subunit precursor of preribosome / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / cytosolic ribosome assembly / ribosomal large subunit biogenesis / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / small-subunit processome / macroautophagy / protein catabolic process / maintenance of translational fidelity / metallopeptidase activity / rRNA processing / protein transport / ribosome biogenesis / 5S rRNA binding / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / protein-macromolecule adaptor activity / cytosolic large ribosomal subunit / nucleic acid binding / cytoplasmic translation / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / GTPase activity / mRNA binding / GTP binding / nucleolus / proteolysis / RNA binding / zinc ion binding / nucleoplasm / metal ion binding / identical protein binding / nucleus / cytosol / cytoplasm Similarity search - Function | ||||||||||||
Biological species | ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.36 Å | ||||||||||||
![]() | Sekulski K / Cruz VE / Weirich CS / Erzberger JP | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 117.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 91.1 KB 91.1 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 11.3 KB | Display | ![]() |
Images | ![]() | 104.8 KB | ||
Filedesc metadata | ![]() | 19.1 KB | ||
Others | ![]() ![]() | 98.4 MB 98.5 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 1.1 MB | Display | ![]() |
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Full document | ![]() | 1.1 MB | Display | |
Data in XML | ![]() | 18.6 KB | Display | |
Data in CIF | ![]() | 24.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7v08MC ![]() 7uooC ![]() 7uqbC ![]() 7uqzC ![]() 7uuiC C: citing same article ( M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a Spb1 D52A suppressor 3 strain | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.08 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: Half-map 1 of nucleoplasmic pre-60S intermediate of the...
File | emd_26941_half_map_1.map | ||||||||||||
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Annotation | Half-map 1 of nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a Spb1 D52A suppressor 3 strain | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map 2 of nucleoplasmic pre-60S intermediate of the...
File | emd_26941_half_map_2.map | ||||||||||||
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Annotation | Half-map 2 of nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state from a Spb1 D52A suppressor 3 strain | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
+Entire : Nucleolar 60S intermediate purified with tags on Tif6 and Nog2.
+Supramolecule #1: Nucleolar 60S intermediate purified with tags on Tif6 and Nog2.
+Macromolecule #1: 60S ribosomal protein L2-A
+Macromolecule #5: rRNA-processing protein
+Macromolecule #7: 60S ribosomal protein L12-A
+Macromolecule #8: Ribosome biogenesis protein ALB1
+Macromolecule #9: 60S ribosomal protein L3
+Macromolecule #10: 60S ribosomal protein L4-A
+Macromolecule #11: 60S ribosomal protein L5
+Macromolecule #12: 60S ribosomal protein L6-A
+Macromolecule #13: 60S ribosomal protein L7-A
+Macromolecule #14: RPL8A isoform 1
+Macromolecule #15: 60S ribosomal protein L9-A
+Macromolecule #16: Bud site selection protein 20
+Macromolecule #17: 60S ribosomal protein L11-A
+Macromolecule #18: Proteasome-interacting protein CIC1
+Macromolecule #19: 60S ribosomal protein L13-A
+Macromolecule #20: 60S ribosomal protein L14-A
+Macromolecule #21: 60S ribosomal protein L15-A
+Macromolecule #22: 60S ribosomal protein L16-A
+Macromolecule #23: 60S ribosomal protein L17-A
+Macromolecule #24: 60S ribosomal protein L18-A
+Macromolecule #25: 60S ribosomal protein L19-A
+Macromolecule #26: 60S ribosomal protein L20-A
+Macromolecule #27: 60S ribosomal protein L21-A
+Macromolecule #28: 60S ribosomal protein L22-A
+Macromolecule #29: 60S ribosomal protein L23-A
+Macromolecule #30: Ribosome assembly factor MRT4
+Macromolecule #31: 60S ribosomal protein L25
+Macromolecule #32: 60S ribosomal protein L26-A
+Macromolecule #33: 60S ribosomal protein L27-A
+Macromolecule #34: 60S ribosomal protein L28
+Macromolecule #35: Nucleolar GTP-binding protein 1
+Macromolecule #36: 60S ribosomal protein L30
+Macromolecule #37: 60S ribosomal protein L31-A
+Macromolecule #38: 60S ribosomal protein L32
+Macromolecule #39: 60S ribosomal protein L33-A
+Macromolecule #40: 60S ribosomal protein L34-A
+Macromolecule #41: 60S ribosomal protein L35-A
+Macromolecule #42: 60S ribosomal protein L36-A
+Macromolecule #43: 60S ribosomal protein L37-A
+Macromolecule #44: RPL38 isoform 1
+Macromolecule #45: 60S ribosomal protein L39
+Macromolecule #46: Nucleolar GTP-binding protein 2
+Macromolecule #47: Pescadillo homolog
+Macromolecule #48: Ribosome biogenesis protein 15
+Macromolecule #49: 60S ribosomal protein L43-A
+Macromolecule #50: Ribosome biogenesis protein NOP53
+Macromolecule #51: Ribosome biogenesis protein NSA2
+Macromolecule #52: Nuclear GTP-binding protein NUG1
+Macromolecule #53: RLP7 isoform 1
+Macromolecule #54: Ribosome biogenesis protein RLP24
+Macromolecule #55: Ribosome production factor 2 homolog
+Macromolecule #56: Ribosome biogenesis regulatory protein
+Macromolecule #57: RSA4 isoform 1
+Macromolecule #58: Eukaryotic translation initiation factor 6
+Macromolecule #59: UPF0642 protein YBL028C
+Macromolecule #60: Probable metalloprotease ARX1
+Macromolecule #2: 25S rRNA
+Macromolecule #3: 5.8S rRNA
+Macromolecule #4: 5S rRNA
+Macromolecule #6: ITS2
+Macromolecule #61: MAGNESIUM ION
+Macromolecule #62: 2-[3-(2-HYDROXY-1,1-DIHYDROXYMETHYL-ETHYLAMINO)-PROPYLAMINO]-2-HY...
+Macromolecule #63: ZINC ION
+Macromolecule #64: GUANOSINE-5'-DIPHOSPHATE
+Macromolecule #65: POTASSIUM ION
+Macromolecule #66: water
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 0.8 mg/mL | |||||||||||||||||||||
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Buffer | pH: 8 Component:
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Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.3 | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 4349 / Average exposure time: 0.05 sec. / Average electron dose: 1.4 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.2 µm / Calibrated defocus min: 0.9 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 81000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |