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- EMDB-26799: Nucleoplasmic pre-60S intermediate of the Nog2 containing post-ro... -

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Basic information

Entry
Database: EMDB / ID: EMD-26799
TitleNucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain
Map dataNucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain
Sample
  • Complex: Map of the nucleolar post-rotation pre-60S intermediate purified with tags on Tif6 and Nog2 from a SPB1 D52A strain.
    • Protein or peptide: Nucleolar GTP-binding protein 2
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Keywordsribosome 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 / GTP binding / nucleolus / nucleoplasm / nucleus
Similarity search - Function
Nucleolar GTP-binding protein 2, N-terminal domain / Nucleolar GTP-binding protein 2 / NGP1NT (NUC091) domain / : / GTP-binding protein, orthogonal bundle domain superfamily / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain / Circularly permuted (CP)-type guanine nucleotide-binding (G) domain profile. / 50S ribosome-binding GTPase / GTP binding domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Nucleolar GTP-binding protein 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae BY4741 (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsSekulski K / Cruz VE / Weirich CS / Erzberger JP
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM135617-01 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR150074 United States
Robert A. Welch FoundationI-1897 United States
CitationJournal: 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
DepositionApr 28, 2022-
Header (metadata) releaseMar 15, 2023-
Map releaseMar 15, 2023-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26799.png

Downloads & links

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Map

FileDownload / File: emd_26799.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å		1.08 Å/pix.
x 400 pix.
= 432. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.0381
Minimum - Maximum-0.091056235 - 0.21717516
Average (Standard dev.)0.00010419026 (±0.010243662)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 432.00003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-map 1 from nucleoplasmic pre-60S intermediate of the...

Fileemd_26799_half_map_1.map
AnnotationHalf-map 1 from nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2 from nucleoplasmic pre-60S intermediate of the...

Fileemd_26799_half_map_2.map
AnnotationHalf-map 2 from nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Map of the nucleolar post-rotation pre-60S intermediate purified ...

EntireName: Map of the nucleolar post-rotation pre-60S intermediate purified with tags on Tif6 and Nog2 from a SPB1 D52A strain.
Components
  • Complex: Map of the nucleolar post-rotation pre-60S intermediate purified with tags on Tif6 and Nog2 from a SPB1 D52A strain.
    • Protein or peptide: Nucleolar GTP-binding protein 2
  • Ligand: MAGNESIUM ION
  • Ligand: POTASSIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Map of the nucleolar post-rotation pre-60S intermediate purified ...

SupramoleculeName: 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
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast)
Molecular weightTheoretical: 3.1 MDa

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Macromolecule #1: Nucleolar GTP-binding protein 2

MacromoleculeName: Nucleolar GTP-binding protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast)
Molecular weightTheoretical: 55.58559 KDa
SequenceString: 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

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #4: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMCH2[CH2NHC(CH2OH)3]2Bis-Tris-Propane
125.0 mMNaClSodium Chloride
25.0 mMKClPotassium Chloride
10.0 mMMgCl2Magnesium Chloride
1.0 mMC9H15O6PHClTCEP
0.01 % (w/v)NP-40
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm 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 beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 925065
Startup modelType of model: EMDB MAP
EMDB ID:

3891

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 50091
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6ylh


Chain - Chain ID: m / Chain - Residue range: 3-469 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-7uui:
Nucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain

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