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Yorodumi- EMDB-26799: Nucleoplasmic pre-60S intermediate of the Nog2 containing post-ro... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26799 | ||||||||||||
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Title | Nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain | ||||||||||||
Map data | Nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain | ||||||||||||
Sample |
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Keywords | ribosome biogenesis / K-loop GTPase / GTPase / ribosome / 5S rRNP | ||||||||||||
Function / homology | Function and homology information regulation of ribosomal subunit export from nucleus / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / large ribosomal subunit rRNA binding / GTPase activity / nucleolus / GTP binding / nucleoplasm / nucleus Similarity search - Function | ||||||||||||
Biological species | Saccharomyces cerevisiae BY4741 (yeast) | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.9 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_26799.map.gz | 228.4 MB | EMDB map data format | |
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Header (meta data) | emd-26799-v30.xml emd-26799.xml | 20.4 KB 20.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_26799_fsc.xml | 14.1 KB | Display | FSC data file |
Images | emd_26799.png | 126.8 KB | ||
Filedesc metadata | emd-26799.cif.gz | 6.2 KB | ||
Others | emd_26799_half_map_1.map.gz emd_26799_half_map_2.map.gz | 194.3 MB 193.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26799 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26799 | HTTPS FTP |
-Validation report
Summary document | emd_26799_validation.pdf.gz | 1.2 MB | Display | EMDB validaton report |
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Full document | emd_26799_full_validation.pdf.gz | 1.2 MB | Display | |
Data in XML | emd_26799_validation.xml.gz | 21.8 KB | Display | |
Data in CIF | emd_26799_validation.cif.gz | 28.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26799 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26799 | HTTPS FTP |
-Related structure data
Related structure data | 7uuiMC 7uooC 7uqbC 7uqzC 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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-Map
File | Download / File: emd_26799.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain | ||||||||||||||||||||
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 from nucleoplasmic pre-60S intermediate of the...
File | emd_26799_half_map_1.map | ||||||||||||
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Annotation | Half-map 1 from nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half-map 2 from nucleoplasmic pre-60S intermediate of the...
File | emd_26799_half_map_2.map | ||||||||||||
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Annotation | Half-map 2 from nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Map of the nucleolar post-rotation pre-60S intermediate purified ...
Entire | Name: Map of the nucleolar post-rotation pre-60S intermediate purified with tags on Tif6 and Nog2 from a SPB1 D52A strain. |
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Components |
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-Supramolecule #1: Map of the nucleolar post-rotation pre-60S intermediate purified ...
Supramolecule | Name: Map of the nucleolar post-rotation pre-60S intermediate purified with tags on Tif6 and Nog2 from a SPB1 D52A strain. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: Saccharomyces cerevisiae BY4741 (yeast) |
Molecular weight | Theoretical: 3.1 MDa |
-Macromolecule #1: Nucleolar GTP-binding protein 2
Macromolecule | Name: Nucleolar GTP-binding protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Saccharomyces cerevisiae BY4741 (yeast) |
Molecular weight | Theoretical: 55.58559 KDa |
Sequence | String: MGTGKKEKSR RIREGDTKDG NLRVKGENFY RDSKRVKFLN MYTSGKEIRN KKGNLIRAAS FQDSTIPDAR VQPDRRWFGN TRVISQDAL QHFRSALGET QKDTYQVLLR RNKLPMSLLE EKDADESPKA RILDTESYAD AFGPKAQRKR PRLAASNLED L VKATNEDI ...String: MGTGKKEKSR RIREGDTKDG NLRVKGENFY RDSKRVKFLN MYTSGKEIRN KKGNLIRAAS FQDSTIPDAR VQPDRRWFGN TRVISQDAL QHFRSALGET QKDTYQVLLR RNKLPMSLLE EKDADESPKA RILDTESYAD AFGPKAQRKR PRLAASNLED L VKATNEDI TKYEEKQVLD ATLGLMGNQE DKENGWTSAA KEAIFSKGQS KRIWNELYKV IDSSDVVIHV LDARDPLGTR CK SVEEYMK KETPHKHLIY VLNKCDLVPT WVAAAWVKHL SKERPTLAFH ASITNSFGKG SLIQLLRQFS QLHTDRKQIS VGF IGYPNT GKSSIINTLR KKKVCQVAPI PGETKVWQYI TLMKRIFLID CPGIVPPSSK DSEEDILFRG VVRVEHVTHP EQYI PGVLK RCQVKHLERT YEISGWKDAT EFIEILARKQ GRLLKGGEPD ESGVSKQILN DFNRGKIPWF VLPPEKEGEE KPKKK EVEK TA UniProtKB: Nucleolar GTP-binding protein 2 |
-Macromolecule #2: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #3: POTASSIUM ION
Macromolecule | Name: POTASSIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: K |
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Molecular weight | Theoretical: 39.098 Da |
-Macromolecule #4: GUANOSINE-5'-DIPHOSPHATE
Macromolecule | Name: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GDP |
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Molecular weight | Theoretical: 443.201 Da |
Chemical component information | ChemComp-GDP: |
-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 - topology: CONTINUOUS / 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: 5434 / 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 |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Chain ID: m / Chain - Residue range: 3-469 / Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Protocol: RIGID BODY FIT |
Output model | PDB-7uui: |