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- EMDB-26637: E.coli RNaseP Holoenzyme with Mg2+ -

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Basic information

Entry
Database: EMDB / ID: EMD-26637
TitleE.coli RNaseP Holoenzyme with Mg2+
Map data
Sample
  • Complex: E.coli RNase P holoenzyme with pre-tRNA substrate AU
    • RNA: E.coli RNase P RNA
    • RNA: Precursor tRNA substrate U(-1) and A(-2)
    • Protein or peptide: Ribonuclease P protein component
  • Ligand: CALCIUM ION
Keywordsribozyme / 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
Ribonuclease P protein component
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsHuang W / Taylor DJ
Funding support United States, 1 items
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, 2022-
Header (metadata) releaseSep 28, 2022-
Map releaseSep 28, 2022-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26637.png

Downloads & links

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Map

FileDownload / File: emd_26637.map.gz / Format: CCP4 / Size: 4.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.49 Å/pix.
x 88 pix.
= 130.926 Å		1.49 Å/pix.
x 100 pix.
= 148.78 Å		1.49 Å/pix.
x 147 pix.
= 218.707 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.4878 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.488
Minimum - Maximum-22.045807 - 26.767612
Average (Standard dev.)-0.000000000002939 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin844886
Dimensions10014788
Spacing88100147
CellA: 130.9264 Å / B: 148.78001 Å / C: 218.70662 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : E.coli RNase P holoenzyme with pre-tRNA substrate AU

EntireName: E.coli RNase P holoenzyme with pre-tRNA substrate AU
Components
  • Complex: E.coli RNase P holoenzyme with pre-tRNA substrate AU
    • RNA: E.coli RNase P RNA
    • RNA: Precursor tRNA substrate U(-1) and A(-2)
    • Protein or peptide: Ribonuclease P protein component
  • Ligand: CALCIUM ION

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Supramolecule #1: E.coli RNase P holoenzyme with pre-tRNA substrate AU

SupramoleculeName: E.coli RNase P holoenzyme with pre-tRNA substrate AU / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: E.coli RNase P RNA

MacromoleculeName: E.coli RNase P RNA / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 121.191891 KDa
SequenceString: GAAGCUGACC AGACAGUCGC CGCUUCGUCG UCGUCCUCUU CGGGGGAGAC GGGCGGAGGG GAGGAAAGUC CGGGCUCCAU AGGGCAGGG UGCCAGGUAA CGCCUGGGGG GGAAACCCAC GACCAGUGCA ACAGAGAGCA AACCGCCGAU GGCCCGCGCA A GCGGGAUC ...String:
GAAGCUGACC AGACAGUCGC CGCUUCGUCG UCGUCCUCUU CGGGGGAGAC GGGCGGAGGG GAGGAAAGUC CGGGCUCCAU AGGGCAGGG UGCCAGGUAA CGCCUGGGGG GGAAACCCAC GACCAGUGCA ACAGAGAGCA AACCGCCGAU GGCCCGCGCA A GCGGGAUC AGGUAAGGGU GAAAGGGUGC GGUAAGAGCG CACCGCGCGG CUGGUAACAG UCCGUGGCAC GGUAAACUCC AC CCGGAGC AAGGCCAAAU AGGGGUUCAU AAGGUACGGC CCGUACUGAA CCCGGGUAGG CUGCUUGAGC CAGUGAGCGA UUG CUGGCC UAGAUGAAUG ACUGUCCACG ACAGAACCCG GCUUAUCGGU CAGUUUC

GENBANK: GENBANK: CP053595.1

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Macromolecule #2: Precursor tRNA substrate U(-1) and A(-2)

MacromoleculeName: Precursor tRNA substrate U(-1) and A(-2) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 26.281574 KDa
SequenceString:
UCUAUGGCUA CGUAGCUCAG UUGGUUAGAG CACAUCACUC AUAAUGAUGG GGUCACAGGU UCGAAUCCCG UCGUAGCCAC CA

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Macromolecule #3: Ribonuclease P protein component

MacromoleculeName: Ribonuclease P protein component / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: ribonuclease P
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 13.10237 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
KLAFPRELRL LTPSQFTFVF QQPQRAGTPQ ITILGRLNSL GHPRIGLTVA KKNVRRAHER NRIKRLTRES FRLRQHELPA MDFVVVAKK GVADLDNRAL SEALEKLWRR HCR

UniProtKB: Ribonuclease P protein component

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Macromolecule #4: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 4 / Number of copies: 10 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.1
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab initio map generated by cryosparc
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 704493
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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