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- PDB-7r6q: State E2 nucleolar 60S ribosome biogenesis intermediate - Foot re... -

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Entry
Database: PDB / ID: 7r6q
TitleState E2 nucleolar 60S ribosome biogenesis intermediate - Foot region model
Components
  • (60S ribosomal protein ...) x 8
  • (Ribosome biogenesis protein ...) x 3
  • 25S rRNA
  • 5.8S rRNA
  • 60S ribosome biogenesis factor Spb1
  • ATP-dependent RNA helicase HAS1
  • ITS-2
  • Nucleolar protein 16
  • Pescadillo homolog
  • Proteasome-interacting protein CIC1
KeywordsRIBOSOME / ribosome biogenesis / DEAD-box ATPase / methyltransferase / nucleolus
Function / homology
Function and homology information


snoRNA release from pre-rRNA / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / PeBoW complex / rRNA primary transcript binding / ATP-dependent activity, acting on RNA / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / proteasome binding / 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 ...snoRNA release from pre-rRNA / exonucleolytic trimming to generate mature 5'-end of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / PeBoW complex / rRNA primary transcript binding / ATP-dependent activity, acting on RNA / maturation of 5.8S rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / proteasome binding / 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 / Nonsense Mediated Decay (NMD) independent of the Exon Junction Complex (EJC) / Nonsense Mediated Decay (NMD) enhanced by the Exon Junction Complex (EJC) / Formation of a pool of free 40S subunits / preribosome, large subunit precursor / L13a-mediated translational silencing of Ceruloplasmin expression / 90S preribosome / ribonucleoprotein complex binding / maturation of LSU-rRNA / proteasome complex / maturation of LSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / ribosomal large subunit biogenesis / maturation of SSU-rRNA from tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / maturation of SSU-rRNA / small-subunit processome / protein catabolic process / rRNA processing / nuclear envelope / ribosomal small subunit biogenesis / ribosomal large subunit assembly / large ribosomal subunit rRNA binding / protein-macromolecule adaptor activity / cytosolic large ribosomal subunit / cytoplasmic translation / RNA helicase activity / rRNA binding / RNA helicase / structural constituent of ribosome / chromatin extrusion motor activity / ATP-dependent H2AZ histone chaperone activity / ATP-dependent H3-H4 histone complex chaperone activity / cohesin loader activity / DNA clamp loader activity / mRNA binding / nucleolus / ATP hydrolysis activity / DNA binding / RNA binding / nucleoplasm / ATP binding / identical protein binding / nucleus / cytosol / cytoplasm
Similarity search - Function
Ribosome biogenesis protein 15, RNA recognition motif / Ribosomal protein L15e / Ribosomal protein L15 / DDX18/Has1, DEAD-box helicase domain / Ribosome biogenesis protein Nop16 / Ribosome biogenesis protein Nop16 / Domain of unknown function DUF4217 / ATP-dependent rRNA helicase SPB4-like, C-terminal extension / DUF4217 / BOP1, N-terminal domain ...Ribosome biogenesis protein 15, RNA recognition motif / Ribosomal protein L15e / Ribosomal protein L15 / DDX18/Has1, DEAD-box helicase domain / Ribosome biogenesis protein Nop16 / Ribosome biogenesis protein Nop16 / Domain of unknown function DUF4217 / ATP-dependent rRNA helicase SPB4-like, C-terminal extension / DUF4217 / BOP1, N-terminal domain / WD repeat BOP1/Erb1 / BOP1NT (NUC169) domain / BOP1NT (NUC169) domain / Pescadillo / Pescadillo N-terminus / BRCT domain / DEAD-box subfamily ATP-dependent helicases signature. / ATP-dependent RNA helicase DEAD-box, conserved site / RNA helicase, DEAD-box type, Q motif / DEAD-box RNA helicase Q motif profile. / breast cancer carboxy-terminal domain / Ribosomal protein L13e, conserved site / Ribosomal protein L13e signature. / Ribosomal protein L13e / Ribosomal protein L13e / Ribosomal protein L23/L25, N-terminal / Ribosomal protein L23, N-terminal domain / BRCT domain profile. / Ribosomal protein L36e signature. / BRCT domain / Ribosomal protein L7A/L8 / Ribosomal protein L1-like / Ribosomal protein L1/ribosomal biogenesis protein / Ribosomal protein L1p/L10e family / BRCT domain superfamily / Ribosomal protein L36e / Ribosomal protein L36e domain superfamily / Ribosomal protein L36e / Ribosomal protein L15e, conserved site / Ribosomal protein L15e signature. / Ribosomal_L15e / Ribosomal protein L15e / Ribosomal protein L15e core domain superfamily / Ribosomal L15 / Ribosomal protein L7, eukaryotic/archaeal / Ribosomal protein L7/L30 / Ribosomal protein L7Ae conserved site / Ribosomal protein L7Ae signature. / DEAD/DEAH box helicase domain / DEAD/DEAH box helicase / Ribosomal protein L7Ae/L8/Nhp2 family / : / RNA recognition motif / RNA recognition motif / Eukaryotic RNA Recognition Motif (RRM) profile. / RNA recognition motif domain / Ribosomal protein L7Ae/L30e/S12e/Gadd45 / Ribosomal protein L7Ae/L30e/S12e/Gadd45 family / RNA-binding domain superfamily / 50S ribosomal protein L30e-like / Helicase conserved C-terminal domain / Ribosomal protein L23/L25, conserved site / Ribosomal protein L23 signature. / Ribosomal protein L30, conserved site / Ribosomal protein L30 signature. / Ribosomal protein L30, ferredoxin-like fold domain / Ribosomal protein L30p/L7e / helicase superfamily c-terminal domain / Ribosomal protein L23 / Ribosomal protein L30, ferredoxin-like fold domain superfamily / Ribosomal protein L25/L23 / Superfamilies 1 and 2 helicase C-terminal domain profile. / Superfamilies 1 and 2 helicase ATP-binding type-1 domain profile. / DEAD-like helicases superfamily / Helicase, C-terminal / Helicase superfamily 1/2, ATP-binding domain / Ribosomal protein L23/L15e core domain superfamily / Nucleotide-binding alpha-beta plait domain superfamily / WD40 repeat, conserved site / Trp-Asp (WD) repeats signature. / Trp-Asp (WD) repeats profile. / Trp-Asp (WD) repeats circular profile. / WD domain, G-beta repeat / WD40 repeats / WD40 repeat / WD40-repeat-containing domain superfamily / WD40/YVTN repeat-like-containing domain superfamily / P-loop containing nucleoside triphosphate hydrolase / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
RNA / RNA (> 10) / RNA (> 100) / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL8A / Proteasome-interacting protein CIC1 / Nucleolar protein 16 / Ribosome biogenesis protein RLP7 ...RNA / RNA (> 10) / RNA (> 100) / Large ribosomal subunit protein uL23 / Large ribosomal subunit protein eL36A / Large ribosomal subunit protein eL15A / Large ribosomal subunit protein eL8A / Proteasome-interacting protein CIC1 / Nucleolar protein 16 / Ribosome biogenesis protein RLP7 / Pescadillo homolog / Ribosome biogenesis protein 15 / ATP-dependent RNA helicase HAS1 / Ribosome biogenesis protein ERB1 / Large ribosomal subunit protein eL13A
Similarity search - Component
Biological speciesSaccharomyces cerevisiae BY4741 (yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 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 23, 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
Revision 1.4Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
6: ITS-2
D: ATP-dependent RNA helicase HAS1
G: 60S ribosomal protein L8-A
K: Proteasome-interacting protein CIC1
n: Pescadillo homolog
o: Ribosome biogenesis protein 15
t: Ribosome biogenesis protein RLP7
X: 60S ribosomal protein L25
m: Ribosome biogenesis protein ERB1
1: 25S rRNA
v: Nucleolar protein 16
2: 5.8S rRNA
N: 60S ribosomal protein L15-A
h: 60S ribosomal protein L35
Z: 60S ribosomal protein L27
g: 60S ribosomal protein L34-A
w: 60S ribosome biogenesis factor Spb1
i: 60S ribosomal protein L36-A
L: 60S ribosomal protein L13-A


Theoretical massNumber of molelcules
Total (without water)610,34919
Polymers610,34919
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area92610 Å2
ΔGint-627 kcal/mol
Surface area163950 Å2

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Components

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RNA chain , 3 types, 3 molecules 612

#1: RNA chain ITS-2


Mass: 27786.307 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#10: RNA chain 25S rRNA


Mass: 95192.117 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#12: RNA chain 5.8S rRNA


Mass: 18223.730 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 , 5 types, 5 molecules DKnvw

#2: Protein ATP-dependent RNA helicase HAS1 / Helicase associated with SET1 protein 1


Mass: 56798.348 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q03532, RNA helicase
#4: Protein Proteasome-interacting protein CIC1 / Core interacting component 1


Mass: 42596.691 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P38779
#5: Protein Pescadillo homolog / Nucleolar protein 7 / Ribosomal RNA-processing protein 13


Mass: 69984.148 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P53261
#11: Protein Nucleolar protein 16


Mass: 26954.447 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P40007
#17: Protein 60S ribosome biogenesis factor Spb1


Mass: 8182.842 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741

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60S ribosomal protein ... , 8 types, 8 molecules GXNhZgiL

#3: Protein 60S ribosomal protein L8-A / L4 / L4-2 / L7a-1 / Large ribosomal subunit protein eL8-A / Maintenance of killer protein 7 / RP6 / YL5


Mass: 28175.820 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P17076
#8: Protein 60S ribosomal protein L25 / Large ribosomal subunit protein uL23 / RP16L / YL25 / YP42'


Mass: 15787.612 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P04456
#13: Protein 60S ribosomal protein L15-A / L13 / Large ribosomal subunit protein eL15-A / RP15R / YL10 / YP18


Mass: 24482.357 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P05748
#14: Protein/peptide 60S ribosomal protein L35


Mass: 2165.599 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#15: Protein/peptide 60S ribosomal protein L27


Mass: 1801.269 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#16: Protein/peptide 60S ribosomal protein L34-A


Mass: 4511.246 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741
#18: Protein 60S ribosomal protein L36-A / L39 / Large ribosomal subunit protein eL36-A / YL39


Mass: 11151.259 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P05745
#19: Protein 60S ribosomal protein L13-A / Large ribosomal subunit protein eL13-A


Mass: 22604.164 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q12690

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

#6: Protein Ribosome biogenesis protein 15 / Nucleolar protein 15


Mass: 25499.186 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P53927
#7: Protein Ribosome biogenesis protein RLP7 / Ribosomal protein L7-like


Mass: 36621.074 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: P40693
#9: Protein Ribosome biogenesis protein ERB1 / Eukaryotic ribosome biogenesis protein 1


Mass: 91830.609 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Saccharomyces cerevisiae BY4741 (yeast) / Strain: BY4741 / References: UniProt: Q04660

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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 ChlorideNaCl1
310 mMMagnesium ChlorideMgCl21
41 mMTCEPC9H15O6PHCl1
50.01 mMNP-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 FIELD / 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: 2.98 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: 32.94 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.002630469
ELECTRON MICROSCOPYf_angle_d0.516143133
ELECTRON MICROSCOPYf_chiral_restr0.03655175
ELECTRON MICROSCOPYf_plane_restr0.00393851
ELECTRON MICROSCOPYf_dihedral_angle_d14.630613070

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