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- PDB-7y18: Crystal structure of ribosomal ITS2 pre-rRNA processing complex f... -

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Basic information

Entry
Database: PDB / ID: 7y18
TitleCrystal structure of ribosomal ITS2 pre-rRNA processing complex from Saccharomyces cerevisiae
Components
  • Polynucleotide 5'-hydroxyl-kinase GRC3
  • Protein LAS1
KeywordsRNA BINDING PROTEIN / RNA processing / Nuclease
Function / homology
Function and homology information


polynucleotide 5'-hydroxyl-kinase activity / Las1 complex / Transferases; Transferring phosphorus-containing groups; Phosphotransferases with an alcohol group as acceptor / nucleolar chromatin / 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) / Major pathway of rRNA processing in the nucleolus and cytosol / termination of RNA polymerase I transcription / preribosome, large subunit precursor / maturation of LSU-rRNA ...polynucleotide 5'-hydroxyl-kinase activity / Las1 complex / Transferases; Transferring phosphorus-containing groups; Phosphotransferases with an alcohol group as acceptor / nucleolar chromatin / 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) / Major pathway of rRNA processing in the nucleolus and cytosol / termination of RNA polymerase I transcription / preribosome, large subunit precursor / maturation of LSU-rRNA / rRNA processing / endonuclease activity / nucleolus / mitochondrion / ATP binding / nucleus / cytoplasm
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
Protein LAS1 / Polynucleotide 5'-hydroxyl-kinase GRC3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae S288C (yeast)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.69 Å
AuthorsChen, J. / Liu, L.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32171286 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
DepositionJun 7, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 29, 2023Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond / pdbx_initial_refinement_model
Revision 1.2Jan 17, 2024Group: 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Polynucleotide 5'-hydroxyl-kinase GRC3
C: Protein LAS1
D: Protein LAS1
B: Polynucleotide 5'-hydroxyl-kinase GRC3
E: Polynucleotide 5'-hydroxyl-kinase GRC3
F: Protein LAS1


Theoretical massNumber of molelcules
Total (without water)395,3336
Polymers395,3336
Non-polymers00
Water3,243180
1
A: Polynucleotide 5'-hydroxyl-kinase GRC3
C: Protein LAS1
D: Protein LAS1
B: Polynucleotide 5'-hydroxyl-kinase GRC3


Theoretical massNumber of molelcules
Total (without water)263,5564
Polymers263,5564
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area22020 Å2
ΔGint-93 kcal/mol
Surface area87910 Å2
MethodPISA
2
E: Polynucleotide 5'-hydroxyl-kinase GRC3
F: Protein LAS1

E: Polynucleotide 5'-hydroxyl-kinase GRC3
F: Protein LAS1


Theoretical massNumber of molelcules
Total (without water)263,5564
Polymers263,5564
Non-polymers00
Water724
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
crystal symmetry operation2_555-x,y,-z1
Buried area19360 Å2
ΔGint-99 kcal/mol
Surface area88080 Å2
MethodPISA
Unit cell
Length a, b, c (Å)233.556, 116.142, 159.311
Angle α, β, γ (deg.)90.000, 96.406, 90.000
Int Tables number5
Space group name H-MC121
Space group name HallC2y
Symmetry operation#1: x,y,z
#2: -x,y,-z
#3: x+1/2,y+1/2,z
#4: -x+1/2,y+1/2,-z
Components on special symmetry positions
IDModelComponents
11F-605-

HOH

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Components

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


Mass: 72342.430 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: GRC3, YLL035W / Production host: Escherichia coli K-12 (bacteria)
References: UniProt: Q07845, Transferases; Transferring phosphorus-containing groups; Phosphotransferases with an alcohol group as acceptor
#2: Protein Protein LAS1


Mass: 59435.332 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae S288C (yeast) / Strain: ATCC 204508 / S288c / Gene: LAS1, YKR063C / Production host: Escherichia coli K-12 (bacteria) / References: UniProt: P36146
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 180 / Source method: isolated from a natural source / Formula: H2O

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 2.72 Å3/Da / Density % sol: 54.71 %
Crystal growTemperature: 289 K / Method: vapor diffusion, hanging drop
Details: 2% tacsimate, pH 7.0, 0.1 M imidazole, pH 7.0, 2% 2-propanole, and 9% PEG 3350

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: SSRF / Beamline: BL19U1 / Wavelength: 0.97853 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jan 13, 2021
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.97853 Å / Relative weight: 1
ReflectionResolution: 3.5→50 Å / Num. obs: 36773 / % possible obs: 98.8 % / Redundancy: 4.5 % / Biso Wilson estimate: 43.42 Å2 / Rpim(I) all: 0.157 / Net I/σ(I): 4.8
Reflection shellResolution: 3.5→3.56 Å / Num. unique obs: 2594 / Rpim(I) all: 0.585

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Processing

Software
NameVersionClassification
PHENIX1.17.1_3660refinement
HKL-30007.21data scaling
HKL-30007.21data reduction
Coot0.7.2.1model building
PHENIX1.17.1_3660phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 6OF4

6of4


Resolution: 3.69→48.63 Å / SU ML: 0.5523 / Cross valid method: FREE R-VALUE / σ(F): 1.37 / Phase error: 31.5715
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
RfactorNum. reflection% reflection
Rfree0.3179 1824 4.96 %
Rwork0.2784 34949 -
obs0.2803 36773 80.12 %
Solvent computationShrinkage radii: 0.9 Å / VDW probe radii: 1.11 Å / Solvent model: FLAT BULK SOLVENT MODEL
Displacement parametersBiso mean: 94.96 Å2
Refinement stepCycle: LAST / Resolution: 3.69→48.63 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms22862 0 0 180 23042
Refine LS restraints
Refine-IDTypeDev idealNumber
X-RAY DIFFRACTIONf_bond_d0.008223433
X-RAY DIFFRACTIONf_angle_d1.490131827
X-RAY DIFFRACTIONf_chiral_restr0.24523509
X-RAY DIFFRACTIONf_plane_restr0.00574047
X-RAY DIFFRACTIONf_dihedral_angle_d29.01338504
LS refinement shell
Resolution (Å)Rfactor RfreeNum. reflection RfreeRfactor RworkNum. reflection RworkRefine-ID% reflection obs (%)
3.69-3.790.926810.381137X-RAY DIFFRACTION1.08
3.79-3.90.355340.2845671X-RAY DIFFRACTION20.13
3.9-4.020.35041100.28011849X-RAY DIFFRACTION55.88
4.03-4.170.32881560.27452779X-RAY DIFFRACTION83.29
4.17-4.340.31161730.27613089X-RAY DIFFRACTION93.09
4.34-4.530.30731530.26813164X-RAY DIFFRACTION94.91
4.53-4.770.31111720.26473284X-RAY DIFFRACTION97.74
4.77-5.070.31971540.26963272X-RAY DIFFRACTION97.75
5.07-5.460.33631810.273315X-RAY DIFFRACTION98.78
5.46-6.010.33371730.3033307X-RAY DIFFRACTION98.86
6.01-6.880.351610.32643372X-RAY DIFFRACTION99.52
6.88-8.660.3061740.28873384X-RAY DIFFRACTION99.8
8.66-48.630.28361820.26373426X-RAY DIFFRACTION99.42

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