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- PDB-7r7c: State E2 nucleolar 60S ribosomal biogenesis intermediate - L1 sta... -

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Basic information

Entry
Database: PDB / ID: 7r7c
TitleState E2 nucleolar 60S ribosomal biogenesis intermediate - L1 stalk local model
Components
  • (60S ribosomal ...) x 2
  • (Ribosome biogenesis protein ...) x 3
  • 25S rRNA
  • 25S rRNA (cytosine(2870)-C(5))-methyltransferase
  • 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase
  • 60S ribosome subunit biogenesis protein NIP7
  • Nucleolar GTP-binding protein 1
  • rRNA-processing protein EBP2
KeywordsRIBOSOME / ribosome biogenesis / DEAD-box ATPases / methyltransferase / nucleolus
Function / homology
Function and homology information


25S rRNA (cytosine2870-C5)-methyltransferase / 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase / rRNA (guanosine-2'-O-)-methyltransferase activity / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA (uridine-2'-O-)-methyltransferase activity / endonucleolytic cleavage in ITS1 upstream of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA (cytosine-C5-)-methyltransferase activity / rRNA (guanine) methyltransferase activity / endonucleolytic cleavage in 5'-ETS of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / intracellular mRNA localization ...25S rRNA (cytosine2870-C5)-methyltransferase / 27S pre-rRNA (guanosine2922-2'-O)-methyltransferase / rRNA (guanosine-2'-O-)-methyltransferase activity / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA (uridine-2'-O-)-methyltransferase activity / endonucleolytic cleavage in ITS1 upstream of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA (cytosine-C5-)-methyltransferase activity / rRNA (guanine) methyltransferase activity / endonucleolytic cleavage in 5'-ETS of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / intracellular mRNA localization / endonucleolytic cleavage to generate mature 5'-end of SSU-rRNA from (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA base methylation / rRNA primary transcript binding / protein folding chaperone complex / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / rRNA methylation / preribosome, large subunit precursor / ribosomal large subunit export from nucleus / mRNA transport / 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) / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of LSU-rRNA / ribosomal large subunit biogenesis / assembly of large subunit precursor of preribosome / ribosomal large subunit assembly / rRNA processing / 5S rRNA binding / negative regulation of translation / ribonucleoprotein complex / mRNA binding / nucleolus / RNA binding / nucleoplasm / identical protein binding / nucleus / cytoplasm
Similarity search - Function
60S ribosomal subunit assembly/export protein Loc1 / Ribosome biogenesis factor, NIP7 / RNA (C5-cytosine) methyltransferase, NOP2 / UPF0113, pre-PUA domain / UPF0113 Pre-PUA domain / UPF0113, PUA domain / UPF0113 PUA domain / Ribosomal RNA small subunit methyltransferase F, N-terminal / N-terminal domain of 16S rRNA methyltransferase RsmF / Nop2p ...60S ribosomal subunit assembly/export protein Loc1 / Ribosome biogenesis factor, NIP7 / RNA (C5-cytosine) methyltransferase, NOP2 / UPF0113, pre-PUA domain / UPF0113 Pre-PUA domain / UPF0113, PUA domain / UPF0113 PUA domain / 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 / Ribosome biogenesis protein BRX1 / : / SAM-dependent methyltransferase RsmB/NOP2-type / RNA (C5-cytosine) methyltransferase / 16S rRNA methyltransferase RsmB/F / SAM-dependent MTase RsmB/NOP-type domain profile. / Putative RNA-binding Domain in PseudoUridine synthase and Archaeosine transglycosylase / PUA domain / PUA domain profile. / PUA domain superfamily / Ribosomal biogenesis NSA2 family / Brix domain / Brix domain / Brix domain profile. / Brix / PUA-like superfamily / Ribosomal RNA methyltransferase, FtsJ domain / FtsJ-like methyltransferase / Ribosomal protein S8e/ribosomal biogenesis NSA2 / Ribosomal protein S8e / S-adenosyl-L-methionine-dependent methyltransferase superfamily
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Ribosome biogenesis protein NSA2 homolog / : / : / 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase / 25S rRNA (cytosine(2870)-C(5))-methyltransferase / 60S ribosomal subunit assembly/export protein LOC1 / Ribosome biogenesis protein BRX1 / 60S ribosome subunit biogenesis protein NIP7
Similarity search - Component
Biological speciesSaccharomyces cerevisiae BY4741 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.71 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 24, 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
l: 60S ribosome subunit biogenesis protein NIP7
m: Ribosome biogenesis protein ERB1
q: 25S rRNA (cytosine(2870)-C(5))-methyltransferase
r: Ribosome biogenesis protein NSA2 homolog
w: 27S pre-rRNA (guanosine(2922)-2'-O)-methyltransferase
7: 60S ribosomal subunit assembly/export protein LOC1
A: Ribosome biogenesis protein BRX1
J: rRNA-processing protein EBP2
b: Nucleolar GTP-binding protein 1
a: 60S ribosomal protein L1-A


Theoretical massNumber of molelcules
Total (without water)282,83411
Polymers282,83411
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area29270 Å2
ΔGint-180 kcal/mol
Surface area98810 Å2

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Components

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Protein , 4 types, 4 molecules lqJb

#2: Protein 60S ribosome subunit biogenesis protein NIP7 / Nuclear import protein 7


Mass: 20411.699 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q08962
#4: Protein 25S rRNA (cytosine(2870)-C(5))-methyltransferase / Nucleolar protein 2


Mass: 69912.164 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
#9: Protein rRNA-processing protein EBP2


Mass: 11444.400 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: A0A7I9FH46
#10: Protein Nucleolar GTP-binding protein 1


Mass: 6401.858 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741

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Ribosome biogenesis protein ... , 3 types, 3 molecules mrA

#3: Protein Ribosome biogenesis protein ERB1 / / Eukaryotic ribosome biogenesis protein 1


Mass: 6833.629 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: A0A6V8S2P3
#5: Protein Ribosome biogenesis protein NSA2 homolog /


Mass: 11673.679 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: A0A6A5PXG0
#8: Protein Ribosome biogenesis protein BRX1 /


Mass: 33585.414 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q08235

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60S ribosomal ... , 2 types, 2 molecules 7a

#7: Protein 60S ribosomal subunit assembly/export protein LOC1 / / Localization of ASH1 mRNA protein 1


Mass: 23652.645 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P43586
#11: Protein 60S ribosomal protein L1-A / Ribosome


Mass: 24538.826 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741

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RNA chain / Protein/peptide , 2 types, 2 molecules 1w

#1: RNA chain 25S rRNA


Mass: 72858.875 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#6: Protein/peptide 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: 1520.849 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

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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: all / 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
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
EM software
IDNameCategory
1RELIONparticle selection
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: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 198000 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 94.05 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002215677
ELECTRON MICROSCOPYf_angle_d0.530522194
ELECTRON MICROSCOPYf_chiral_restr0.03532688
ELECTRON MICROSCOPYf_plane_restr0.00351989
ELECTRON MICROSCOPYf_dihedral_angle_d15.84146666

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