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- PDB-7uui: Nucleoplasmic pre-60S intermediate of the Nog2 containing post-ro... -

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Basic information

Entry
Database: PDB / ID: 7uui
TitleNucleoplasmic pre-60S intermediate of the Nog2 containing post-rotation state from a SPB1 D52A strain
ComponentsNucleolar GTP-binding protein 2
KeywordsRIBOSOME / ribosome biogenesis / K-loop GTPase / GTPase / 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
GUANOSINE-5'-DIPHOSPHATE / : / Nucleolar GTP-binding protein 2
Similarity search - Component
Biological speciesSaccharomyces cerevisiae BY4741 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsSekulski, K. / Cruz, V.E. / Weirich, C.S. / Erzberger, J.P.
Funding support United States, 3items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 15, 2023Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / em_3d_fitting_list
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
m: Nucleolar GTP-binding protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)56,0924
Polymers55,5861
Non-polymers5073
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Nucleolar GTP-binding protein 2


Mass: 55585.590 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / References: UniProt: P53742
#2: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#3: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-GDP / GUANOSINE-5'-DIPHOSPHATE


Type: RNA linking / Mass: 443.201 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H15N5O11P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: GDP, energy-carrying molecule*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Map of the nucleolar post-rotation pre-60S intermediate purified with tags on Tif6 and Nog2 from a SPB1 D52A strain.
Type: RIBOSOME / Entity ID: #1 / Source: NATURAL
Molecular weightValue: 3.1 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae BY4741 (yeast)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMBis-Tris-PropaneCH2[CH2NHC(CH2OH)3]21
2125 mMSodium ChlorideNaCl1
325 mMPotassium ChlorideKCl1
410 mMMagnesium ChlorideMgCl21
51 mMTCEPC9H15O6PHCl1
60.01 % (w/v)NP-401
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 0.05 sec. / Electron dose: 1.4 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5434

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameCategory
4GctfCTF correction
9RELIONinitial Euler assignment
CTF correctionType: NONE
Particle selectionNum. of particles selected: 925065
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 50091 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6YLH

6ylh


Pdb chain-ID: m / Accession code: 6YLH / Pdb chain residue range: 3-469 / Source name: PDB / Type: experimental model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 21.66 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00253745
ELECTRON MICROSCOPYf_angle_d0.50385049
ELECTRON MICROSCOPYf_chiral_restr0.0404550
ELECTRON MICROSCOPYf_plane_restr0.0036641
ELECTRON MICROSCOPYf_dihedral_angle_d14.42141448

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