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- PDB-8j60: Structural and mechanistic insight into ribosomal ITS2 RNA proces... -

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Basic information

Entry
Database: PDB / ID: 8j60
TitleStructural and mechanistic insight into ribosomal ITS2 RNA processing by nuclease-kinase machinery
Components
  • LAS1 protein
  • Polynucleotide 5'-hydroxyl-kinase GRC3
KeywordsRNA BINDING PROTEIN / endoribonuclease / ribosome biosynthesis / Las1 / Grc3
Function / homology
Function and homology information


polynucleotide 5'-hydroxyl-kinase activity / Las1 complex / maturation of 5.8S rRNA / cleavage in ITS2 between 5.8S rRNA and LSU-rRNA of tricistronic rRNA transcript (SSU-rRNA, 5.8S rRNA, LSU-rRNA) / preribosome, large subunit precursor / maturation of LSU-rRNA / endonuclease activity / ATP binding / nucleus
Similarity search - Function
Las1 / Las1-like / Polyribonucleotide 5'-hydroxyl-kinase Clp1, P-loop domain / Polyribonucleotide 5-hydroxyl-kinase Clp1/Grc3 / mRNA cleavage and polyadenylation factor CLP1 P-loop / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Polynucleotide 5'-hydroxyl-kinase GRC3 / LAS1 protein
Similarity search - Component
Biological speciesCyberlindnera jadinii (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsChen, J. / Chen, H. / Li, S. / Lin, X. / Hu, R. / Zhang, K. / Liu, L.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: Elife / Year: 2024
Title: Structural and mechanistic insights into ribosomal ITS2 RNA processing by nuclease-kinase machinery.
Authors: Jiyun Chen / Hong Chen / Shanshan Li / Xiaofeng Lin / Rong Hu / Kaiming Zhang / Liang Liu /
Abstract: Precursor ribosomal RNA (pre-rRNA) processing is a key step in ribosome biosynthesis and involves numerous RNases. A HEPN (higher eukaryote and prokaryote nucleotide binding) nuclease Las1 and a ...Precursor ribosomal RNA (pre-rRNA) processing is a key step in ribosome biosynthesis and involves numerous RNases. A HEPN (higher eukaryote and prokaryote nucleotide binding) nuclease Las1 and a polynucleotide kinase Grc3 assemble into a tetramerase responsible for rRNA maturation. Here, we report the structures of full-length and Las1-Grc3 complexes, and Las1. The Las1-Grc3 structures show that the central coiled-coil domain of Las1 facilitates pre-rRNA binding and cleavage, while the Grc3 C-terminal loop motif directly binds to the HEPN active center of Las1 and regulates pre-rRNA cleavage. Structural comparison between Las1 and Las1-Grc3 complex exhibits that Grc3 binding induces conformational rearrangements of catalytic residues associated with HEPN nuclease activation. Biochemical assays identify that Las1 processes pre-rRNA at the two specific sites (C2 and C2'), which greatly facilitates rRNA maturation. Our structures and specific pre-rRNA cleavage findings provide crucial insights into the mechanism and pathway of pre-rRNA processing in ribosome biosynthesis.
History
DepositionApr 24, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 17, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 16, 2024Group: Structure summary / Category: pdbx_entry_details / pdbx_modification_feature
Revision 1.2Jun 18, 2025Group: Data collection / Category: em_software / Item: _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Polynucleotide 5'-hydroxyl-kinase GRC3
C: LAS1 protein
D: LAS1 protein
A: Polynucleotide 5'-hydroxyl-kinase GRC3


Theoretical massNumber of molelcules
Total (without water)238,0534
Polymers238,0534
Non-polymers00
Water00
1


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

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Components

#1: Protein Polynucleotide 5'-hydroxyl-kinase GRC3 / Polynucleotide 5'-hydroxyl-kinase grc3


Mass: 69612.867 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cyberlindnera jadinii (fungus) / Gene: GRC3, BN1211_2636, CYBJADRAFT_171070
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0H5C3P3
#2: Protein LAS1 protein


Mass: 49413.621 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Cyberlindnera jadinii (fungus) / Gene: LAS1, BN1211_1791
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: A0A0H5CBH3
Has protein modificationY

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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: CjLas1-Grc3 complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Cyberlindnera jadinii (fungus)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 3000 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 523843 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410232
ELECTRON MICROSCOPYf_angle_d0.70413936
ELECTRON MICROSCOPYf_dihedral_angle_d5.4191395
ELECTRON MICROSCOPYf_chiral_restr0.0441645
ELECTRON MICROSCOPYf_plane_restr0.0071765

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