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- PDB-7naf: State E2 nucleolar 60S ribosomal biogenesis intermediate - Spb1-M... -

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Basic information

Entry
Database: PDB / ID: 7naf
TitleState E2 nucleolar 60S ribosomal biogenesis intermediate - Spb1-MTD local model
Components
  • (60S ribosomal protein ...) x 5
  • (60S ribosome ...) x 2
  • 25S rRNA
  • 25S rRNA (cytosine(2870)-C(5))-methyltransferase
  • 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase
  • Eukaryotic translation initiation factor 6
  • Nuclear GTP-binding protein NUG1
  • Nucleolar complex protein 2
  • Ribosomal RNA-processing protein 17
  • Ribosome biogenesis protein RLP24
  • UPF0642 protein YBL028C
KeywordsRIBOSOME / ribosome biogenesis / DEAD-box ATPases / methyltransferase / nucleolus
Function / homology
Function and homology information


25S rRNA (cytosine2870-C5)-methyltransferase / Noc1p-Noc2p complex / 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase / Noc2p-Noc3p complex / rRNA (guanosine-2'-O-)-methyltransferase activity / rRNA (uridine-2'-O-)-methyltransferase activity / rRNA (cytosine-C5-)-methyltransferase activity / rRNA (guanine) methyltransferase activity / rRNA base methylation / positive regulation of ATP-dependent activity ...25S rRNA (cytosine2870-C5)-methyltransferase / Noc1p-Noc2p complex / 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase / Noc2p-Noc3p complex / rRNA (guanosine-2'-O-)-methyltransferase activity / rRNA (uridine-2'-O-)-methyltransferase activity / rRNA (cytosine-C5-)-methyltransferase activity / rRNA (guanine) methyltransferase activity / rRNA base methylation / positive regulation of ATP-dependent activity / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA methylation / maturation of 5.8S rRNA / ribosomal subunit export from nucleus / 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 / ATPase activator activity / Formation of a pool of free 40S subunits / 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) / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / ribosomal large subunit export from nucleus / regulation of translational fidelity / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA / ribosomal large subunit biogenesis / translation initiation factor activity / assembly of large subunit precursor of preribosome / cytosolic ribosome assembly / maintenance of translational fidelity / ribosomal large subunit assembly / rRNA processing / large ribosomal subunit rRNA binding / protein transport / cytoplasmic translation / cytosolic large ribosomal subunit / ATPase binding / structural constituent of ribosome / translation / mRNA binding / GTPase activity / GTP binding / nucleolus / mitochondrion / RNA binding / nucleoplasm / nucleus / cytosol / cytoplasm
Similarity search - Function
Nucleolar complex protein 2 / Noc2p family / RNA (C5-cytosine) methyltransferase, NOP2 / Ribosomal RNA small subunit methyltransferase F, N-terminal / N-terminal domain of 16S rRNA methyltransferase RsmF / Nop2p / Bacterial Fmu (Sun)/eukaryotic nucleolar NOL1/Nop2p, conserved site / NOL1/NOP2/sun family signature. / Ribosomal RNA methyltransferase, SPB1-like, C-terminal / Ribosomal RNA methyltransferase Spb1, domain of unknown function DUF3381 ...Nucleolar complex protein 2 / Noc2p family / RNA (C5-cytosine) methyltransferase, NOP2 / Ribosomal RNA small subunit methyltransferase F, N-terminal / N-terminal domain of 16S rRNA methyltransferase RsmF / Nop2p / Bacterial Fmu (Sun)/eukaryotic nucleolar NOL1/Nop2p, conserved site / NOL1/NOP2/sun family signature. / Ribosomal RNA methyltransferase, SPB1-like, C-terminal / Ribosomal RNA methyltransferase Spb1, domain of unknown function DUF3381 / AdoMet-dependent rRNA methyltransferase SPB1-like / Spb1 C-terminal domain / Ribosomal RNA methyltransferase Spb1, DUF3381 / Ribosomal RNA large subunit methyltransferase E / : / Domain of unknown function DUF2423 / Protein of unknown function (DUF2423) / SAM-dependent methyltransferase RsmB/NOP2-type / RNA (C5-cytosine) methyltransferase / 16S rRNA methyltransferase RsmB/F / SAM-dependent MTase RsmB/NOP-type domain profile. / Guanine nucleotide-binding protein-like 3, N-terminal domain / GNL3L/Grn1 putative GTPase / 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. / Translation initiation factor IF6 / eIF-6 family / translation initiation factor 6 / 50S ribosome-binding GTPase / GTP binding domain / metallochaperone-like domain / TRASH domain / Ribosomal protein L31e, conserved site / Ribosomal protein L31e / Ribosomal protein L31e domain superfamily / Ribosomal_L31e / Ribosomal RNA methyltransferase, FtsJ domain / FtsJ-like methyltransferase / Ribosomal protein L3, domain 3, archaeal type superfamily / Ribosomal protein L3, archaeal/eukaryotic type / Ribosomal protein L31e / Ribosomal protein L24e-related / Ribosomal protein L24e/L24 superfamily / Ribosomal protein L24e / Ribosomal protein L31e signature. / Ribosomal protein L14P, conserved site / Ribosomal protein L14 signature. / Ribosomal protein L14p/L23e / Ribosomal protein L14P / Ribosomal protein L14 superfamily / Ribosomal protein L3, conserved site / Ribosomal protein L14p/L23e / Ribosomal protein L3 / Ribosomal protein L3 / Ribosomal protein L3 signature. / Translation protein, beta-barrel domain superfamily / S-adenosyl-L-methionine-dependent methyltransferase superfamily / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
S-ADENOSYL-L-HOMOCYSTEINE / RNA / RNA (> 10) / RNA (> 100) / Large ribosomal subunit protein eL31A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL3 / 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase / UPF0642 protein YBL028C / Nucleolar complex protein 2 ...S-ADENOSYL-L-HOMOCYSTEINE / RNA / RNA (> 10) / RNA (> 100) / Large ribosomal subunit protein eL31A / Large ribosomal subunit protein uL14A / Large ribosomal subunit protein uL3 / 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase / UPF0642 protein YBL028C / Nucleolar complex protein 2 / Nuclear GTP-binding protein NUG1 / 25S rRNA (cytosine(2870)-C(5))-methyltransferase / Ribosome biogenesis protein RLP24 / Eukaryotic translation initiation factor 6
Similarity search - Component
Biological speciesSaccharomyces cerevisiae BY4741 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.13 Å
AuthorsCruz, V.E. / Sekulski, K. / Peddada, N. / 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 Struct Mol Biol / Year: 2022
Title: Sequence-specific remodeling of a topologically complex RNP substrate by Spb4.
Authors: Victor Emmanuel Cruz / Kamil Sekulski / Nagesh Peddada / Carolin Sailer / Sahana Balasubramanian / Christine S Weirich / Florian Stengel / Jan P Erzberger /
Abstract: DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes are at odds with their complex cellular ...DEAD-box ATPases are ubiquitous enzymes essential in all aspects of RNA biology. However, the limited in vitro catalytic activities described for these enzymes are at odds with their complex cellular roles, most notably in driving large-scale RNA remodeling steps during the assembly of ribonucleoproteins (RNPs). We describe cryo-EM structures of 60S ribosomal biogenesis intermediates that reveal how context-specific RNA unwinding by the DEAD-box ATPase Spb4 results in extensive, sequence-specific remodeling of rRNA secondary structure. Multiple cis and trans interactions stabilize Spb4 in a post-catalytic, high-energy intermediate that drives the organization of the three-way junction at the base of rRNA domain IV. This mechanism explains how limited strand separation by DEAD-box ATPases is leveraged to provide non-equilibrium directionality and ensure efficient and accurate RNP assembly.
History
DepositionJun 21, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 9, 2022Provider: repository / Type: Initial release
Revision 1.1Dec 14, 2022Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Dec 21, 2022Group: Database references / Category: citation / citation_author
Item: _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.3Dec 28, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
1: 25S rRNA
5: Ribosomal RNA-processing protein 17
8: Nucleolar complex protein 2
V: 60S ribosomal protein L23-A
u: Ribosome biogenesis protein RLP24
w: 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase
b: 60S ribosome biogenesis factor Nog1
d: 60S ribosomal protein L31-A
U: 60S ribosomal protein L22-A
R: 60S ribosome ribosomal protein L19A
c: 60S ribosomal protein L30
B: 60S ribosomal protein L3
z: UPF0642 protein YBL028C
s: Nuclear GTP-binding protein NUG1
y: Eukaryotic translation initiation factor 6
q: 25S rRNA (cytosine(2870)-C(5))-methyltransferase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)311,69117
Polymers311,30716
Non-polymers3841
Water362
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area52520 Å2
ΔGint-359 kcal/mol
Surface area118940 Å2

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Components

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RNA chain , 1 types, 1 molecules 1

#1: RNA chain 25S rRNA


Mass: 121655.477 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741

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Protein , 4 types, 4 molecules 5uwy

#2: Protein Ribosomal RNA-processing protein 17


Mass: 13077.044 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#5: Protein Ribosome biogenesis protein RLP24 / / Ribosomal protein L24-like


Mass: 7786.305 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q07915
#6: Protein 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase / 2'-O-ribose RNA methyltransferase SPB1 / AdoMet-dependent rRNA methyltransferase SPB1 / S-adenosyl- ...2'-O-ribose RNA methyltransferase SPB1 / AdoMet-dependent rRNA methyltransferase SPB1 / S-adenosyl-L-methionine-dependent methyltransferase / Suppressor of PAB1 protein 1


Mass: 45405.633 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
References: UniProt: P25582, 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase
#15: Protein Eukaryotic translation initiation factor 6 / eIF-6


Mass: 26476.605 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q12522

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Protein/peptide , 4 types, 4 molecules 8zsq

#3: Protein/peptide Nucleolar complex protein 2


Mass: 4749.660 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P39744
#13: Protein/peptide UPF0642 protein YBL028C


Mass: 646.780 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P38202
#14: Protein/peptide Nuclear GTP-binding protein NUG1 / Nuclear GTPase 1


Mass: 977.232 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P40010
#16: Protein/peptide 25S rRNA (cytosine(2870)-C(5))-methyltransferase / Nucleolar protein 2


Mass: 3821.383 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
References: UniProt: P40991, 25S rRNA (cytosine2870-C5)-methyltransferase

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60S ribosomal protein ... , 5 types, 5 molecules VdUcB

#4: Protein 60S ribosomal protein L23-A / Ribosome / L17a / Large ribosomal subunit protein uL14-A / YL32


Mass: 14218.625 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P0CX41
#8: Protein 60S ribosomal protein L31-A / Ribosome / L34 / Large ribosomal subunit protein eL31-A / YL28


Mass: 7025.393 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P0C2H8
#9: Protein/peptide 60S ribosomal protein L22-A / Ribosome


Mass: 2350.864 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#11: Protein/peptide 60S ribosomal protein L30 /


Mass: 4390.093 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#12: Protein 60S ribosomal protein L3 / / Large ribosomal subunit protein uL3 / Maintenance of killer protein 8 / RP1 / Trichodermin ...Large ribosomal subunit protein uL3 / Maintenance of killer protein 8 / RP1 / Trichodermin resistance protein / YL1


Mass: 42452.184 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P14126

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60S ribosome ... , 2 types, 2 molecules bR

#7: Protein 60S ribosome biogenesis factor Nog1


Mass: 8783.554 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
References: 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase
#10: Protein 60S ribosome ribosomal protein L19A


Mass: 7490.019 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741

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Non-polymers , 2 types, 3 molecules

#17: Chemical ChemComp-SAH / S-ADENOSYL-L-HOMOCYSTEINE / S-Adenosyl-L-homocysteine


Type: L-peptide linking / Mass: 384.411 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C14H20N6O5S / Feature type: SUBJECT OF INVESTIGATION
#18: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O

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Details

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: Nucleolar 60S intermediate purified with tags on Ytm1 and Spb4.
Type: RIBOSOME / Entity ID: #1-#16 / Source: NATURAL
Molecular weightValue: 3.3 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
2150 mMSodium ChlorideNaClSodium chloride1
310 mMMagnesium ChlorideMgCl21
41 mMTCEPC9H15O6PHCl1
50.01 % (w/v)NP-401
SpecimenConc.: 0.45 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 R2/2
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 FIELDBright-field microscopy / Nominal magnification: 81000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 900 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: 1.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4523

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameCategory
2SerialEMimage acquisition
4GctfCTF correction
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
12RELIONclassification
13RELION3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 825096
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.13 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 54.46 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002319938
ELECTRON MICROSCOPYf_angle_d0.458928556
ELECTRON MICROSCOPYf_chiral_restr0.03263503
ELECTRON MICROSCOPYf_plane_restr0.00312221
ELECTRON MICROSCOPYf_dihedral_angle_d14.19145902

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