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- PDB-7uo1: E.coli RNaseP Holoenzyme with Mg2+ -

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Basic information

Entry
Database: PDB / ID: 7uo1
TitleE.coli RNaseP Holoenzyme with Mg2+
Components
  • E.coli RNase P RNA
  • Precursor tRNA substrate U(-1) and A(-2)
  • Ribonuclease P protein component
KeywordsHYDROLASE/RNA / ribozyme / protein-RNA complex / divalent ion / RNA / HYDROLASE-RNA complex
Function / homology
Function and homology information


3'-tRNA processing endoribonuclease activity / ribonuclease P complex / ribonuclease P / ribonuclease P activity / tRNA 5'-leader removal / tRNA binding
Similarity search - Function
Ribonuclease P / Ribonuclease P, conserved site / Ribonuclease P / Bacterial ribonuclease P protein component signature. / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA (> 100) / Ribonuclease P protein component
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHuang, W. / Taylor, D.J.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM133841 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structural and mechanistic basis for recognition of alternative tRNA precursor substrates by bacterial ribonuclease P.
Authors: Jiaqiang Zhu / Wei Huang / Jing Zhao / Loc Huynh / Derek J Taylor / Michael E Harris /
Abstract: Binding of precursor tRNAs (ptRNAs) by bacterial ribonuclease P (RNase P) involves an encounter complex (ES) that isomerizes to a catalytic conformation (ES*). However, the structures of ...Binding of precursor tRNAs (ptRNAs) by bacterial ribonuclease P (RNase P) involves an encounter complex (ES) that isomerizes to a catalytic conformation (ES*). However, the structures of intermediates and the conformational changes that occur during binding are poorly understood. Here, we show that pairing between the 5' leader and 3'RCCA extending the acceptor stem of ptRNA inhibits ES* formation. Cryo-electron microscopy single particle analysis reveals a dynamic enzyme that becomes ordered upon formation of ES* in which extended acceptor stem pairing is unwound. Comparisons of structures with alternative ptRNAs reveals that once unwinding is completed RNase P primarily uses stacking interactions and shape complementarity to accommodate alternative sequences at its cleavage site. Our study reveals active site interactions and conformational changes that drive molecular recognition by RNase P and lays the foundation for understanding how binding interactions are linked to helix unwinding and catalysis.
History
DepositionApr 12, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 28, 2022Provider: repository / Type: Initial release
Revision 1.1Jun 12, 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
B: E.coli RNase P RNA
C: Precursor tRNA substrate U(-1) and A(-2)
A: Ribonuclease P protein component
hetero molecules


Theoretical massNumber of molelcules
Total (without water)160,97713
Polymers160,5763
Non-polymers40110
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, C1
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain E.coli RNase P RNA


Mass: 121191.891 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Production host: in vitro transcription vector pT7-TP(deltai) (others)
References: GenBank: 1845291569
#2: RNA chain Precursor tRNA substrate U(-1) and A(-2)


Mass: 26281.574 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli)
Production host: in vitro transcription vector pT7-TP(deltai) (others)
#3: Protein Ribonuclease P protein component / RNase P protein / RNaseP protein / Protein C5


Mass: 13102.370 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Production host: Escherichia coli (E. coli) / References: UniProt: C3SLK7, ribonuclease P
#4: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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: E.coli RNase P holoenzyme with pre-tRNA substrate AU / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.1
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: 1000 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 704493 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00511867
ELECTRON MICROSCOPYf_angle_d1.07618036
ELECTRON MICROSCOPYf_dihedral_angle_d26.6165581
ELECTRON MICROSCOPYf_chiral_restr0.0592141
ELECTRON MICROSCOPYf_plane_restr0.006619

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