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- PDB-6of3: Precursor ribosomal RNA processing complex, State 1. -

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Basic information

Entry
Database: PDB / ID: 6of3
TitlePrecursor ribosomal RNA processing complex, State 1.
Components
  • CLP1_P domain-containing protein
  • Ribonuclease
KeywordsRNA BINDING PROTEIN / Complex / Ribonuclease / Polynucleotide kinase
Function / homologyLas1 / Polyribonucleotide 5'-hydroxyl-kinase Clp1, P-loop domain / Las1-like / mRNA cleavage and polyadenylation factor CLP1 P-loop / Las1 complex / rRNA processing / endonuclease activity / CLP1_P domain-containing protein / Uncharacterized protein
Function and homology information
Biological speciesChaetomium thermophilum (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsPillon, M.C. / Hsu, A.L. / Krahn, J.M. / Williams, J.G. / Goslen, K.H. / Sobhany, M. / Borgnia, M.J. / Stanley, R.E.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Environmental Health SciencesZIA ES103247 United States
CitationJournal: Nat. Struct. Mol. Biol. / Year: 2019
Title: Cryo-EM reveals active site coordination within a multienzyme pre-rRNA processing complex.
Authors: Monica C Pillon / Allen L Hsu / Juno M Krahn / Jason G Williams / Kevin H Goslen / Mack Sobhany / Mario J Borgnia / Robin E Stanley /
Abstract: Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The ...Ribosome assembly is a complex process reliant on the coordination of trans-acting enzymes to produce functional ribosomal subunits and secure the translational capacity of cells. The endoribonuclease (RNase) Las1 and the polynucleotide kinase (PNK) Grc3 assemble into a multienzyme complex, herein designated RNase PNK, to orchestrate processing of precursor ribosomal RNA (rRNA). RNase PNK belongs to the functionally diverse HEPN nuclease superfamily, whose members rely on distinct cues for nuclease activation. To establish how RNase PNK coordinates its dual enzymatic activities, we solved a series of cryo-EM structures of Chaetomium thermophilum RNase PNK in multiple conformational states. The structures reveal that RNase PNK adopts a butterfly-like architecture, harboring a composite HEPN nuclease active site flanked by discrete RNA kinase sites. We identify two molecular switches that coordinate nuclease and kinase function. Together, our structures and corresponding functional studies establish a new mechanism of HEPN nuclease activation essential for ribosome production.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMar 28, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Ribonuclease
B: CLP1_P domain-containing protein
D: Ribonuclease
E: CLP1_P domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)228,1028
Polymers227,0074
Non-polymers1,0954
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide Ribonuclease /


Mass: 43842.871 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0066080 / Production host: Escherichia coli (E. coli) / References: UniProt: G0SGE9
#2: Protein/peptide CLP1_P domain-containing protein


Mass: 69660.539 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chaetomium thermophilum (strain DSM 1495 / CBS 144.50 / IMI 039719) (fungus)
Strain: DSM 1495 / CBS 144.50 / IMI 039719 / Gene: CTHT_0016120 / Production host: Escherichia coli (E. coli) / References: UniProt: G0S263
#3: Chemical ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O12P3S
Comment: ATP-gamma-S (energy-carrying molecule analogue) *YM
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Magnesium
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: Precursor Ribosomal RNA Processing Complex / Type: COMPLEX
Details: Precursor Ribosomal RNA Processing Complex coordinates removal of a transcribed spacer.
Entity ID: 1, 2 / Source: RECOMBINANT
Molecular weightValue: 0.234 MDa / Experimental value: NO
Source (natural)Organism: Chaetomium thermophilum (fungus)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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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
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 74582 / Symmetry type: POINT
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00811400
f_angle_d1.5715574
f_dihedral_angle_d11.66796
f_chiral_restr0.0751742
f_plane_restr0.0111990

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