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Yorodumi- EMDB-26651: Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rot... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26651 | ||||||||||||
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Title | Nucleoplasmic pre-60S intermediate of the Nog2 containing pre-rotation state | ||||||||||||
Map data | Nog2-containing pre-60S intermediate in the pre-rotation state | ||||||||||||
Sample |
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Keywords | ribosome biogenesis / K-loop GTPase / GTPase / RIBOSOME | ||||||||||||
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) / hexon binding / pre-mRNA 5'-splice site binding ...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) / hexon binding / 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) / Formation of a pool of free 40S subunits / negative regulation of mRNA splicing, via spliceosome / ATPase activator activity / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / translational elongation / ribosomal large subunit export from nucleus / regulation of translational fidelity / protein-RNA complex assembly / ribonucleoprotein complex binding / ribosomal subunit export from nucleus / 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 / maturation of LSU-rRNA / translation initiation factor activity / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / proteasome complex / ribosomal large subunit biogenesis / 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 / macroautophagy / maintenance of translational fidelity / protein catabolic process / rRNA processing / metallopeptidase activity / protein transport / ribosome biogenesis / viral capsid / ribosomal large subunit assembly / protein-macromolecule adaptor activity / ATPase binding / large ribosomal subunit rRNA binding / 5S rRNA binding / cytoplasmic translation / cytosolic large ribosomal subunit / nucleic acid binding / negative regulation of translation / rRNA binding / ribosome / structural constituent of ribosome / translation / GTPase activity / mRNA binding / GTP binding / host cell nucleus / nucleolus / 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 | single particle reconstruction / cryo EM / Resolution: 2.34 Å | ||||||||||||
Authors | Sekulski K / Cruz VE / Weirich CS / Erzberger JP | ||||||||||||
Funding support | United States, 3 items
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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
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26651.map.gz | 229 MB | EMDB map data format | |
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Header (meta data) | emd-26651-v30.xml emd-26651.xml | 91.1 KB 91.1 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_26651_fsc.xml | 14 KB | Display | FSC data file |
Images | emd_26651.png | 93.6 KB | ||
Masks | emd_26651_msk_1.map | 244.1 MB | Mask map | |
Filedesc metadata | emd-26651.cif.gz | 19.2 KB | ||
Others | emd_26651_half_map_1.map.gz emd_26651_half_map_2.map.gz | 193.5 MB 193.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26651 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26651 | HTTPS FTP |
-Validation report
Summary document | emd_26651_validation.pdf.gz | 1019 KB | Display | EMDB validaton report |
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Full document | emd_26651_full_validation.pdf.gz | 1018.6 KB | Display | |
Data in XML | emd_26651_validation.xml.gz | 21.4 KB | Display | |
Data in CIF | emd_26651_validation.cif.gz | 28.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26651 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26651 | HTTPS FTP |
-Related structure data
Related structure data | 7uooMC 7uqbC 7uqzC 7uuiC 7v08C C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_26651.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | Nog2-containing pre-60S intermediate in the pre-rotation state | ||||||||||||||||||||||||||||||||||||
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
-Mask #1
File | emd_26651_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: Half-map 1 of the Nog2-containing pre-60S intermediate in...
File | emd_26651_half_map_1.map | ||||||||||||
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Annotation | Half-map 1 of the Nog2-containing pre-60S intermediate in the pre-rotation state | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map 2 of the Nog2-containing pre-60S intermediate in...
File | emd_26651_half_map_2.map | ||||||||||||
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Annotation | Half-map 2 of the Nog2-containing pre-60S intermediate in the pre-rotation state | ||||||||||||
Projections & Slices |
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Density Histograms |
-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: 60S ribosomal protein L8-A
+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: 60S ribosomal protein L38
+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: Ribosome biogenesis protein RLP7
+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 rRNA
+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: GUANOSINE-5'-TRIPHOSPHATE
+Macromolecule #66: POTASSIUM ION
+Macromolecule #67: water
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-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 / Pretreatment - Type: GLOW DISCHARGE | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-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: 5580 / Average exposure time: 0.05 sec. / Average electron dose: 1.4 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
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 |