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- PDB-7f3e: Cryo-EM structure of the minimal protein-only RNase P from Aquife... -

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Basic information

Entry
Database: PDB / ID: 7f3e
TitleCryo-EM structure of the minimal protein-only RNase P from Aquifex aeolicus
ComponentsRNA-free ribonuclease P
KeywordsRNA BINDING PROTEIN / Molecular evolution / RNase P / pre-tRNA / cryo-EM / dodecamer
Function / homologyRNA-free ribonuclease P / PINc domain ribonuclease / ribonuclease P / ribonuclease P activity / tRNA 5'-leader removal / PIN-like domain superfamily / RNA-free ribonuclease P
Function and homology information
Biological speciesAquifex aeolicus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsTeramoto, T. / Koyasu, T. / Adachi, N. / Kawasaki, M. / Moriya, T. / Numata, T. / Senda, T. / Kakuta, Y.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP21am0101071 Japan
Japan Society for the Promotion of Science (JSPS)21K06032 Japan
Japan Society for the Promotion of Science (JSPS)JP18J00191 Japan
CitationJournal: J Biol Chem / Year: 2021
Title: Minimal protein-only RNase P structure reveals insights into tRNA precursor recognition and catalysis.
Authors: Takamasa Teramoto / Takeshi Koyasu / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / Tomoyuki Numata / Toshiya Senda / Yoshimitsu Kakuta /
Abstract: Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in ...Ribonuclease P (RNase P) is an endoribonuclease that catalyzes the processing of the 5' leader sequence of precursor tRNA (pre-tRNA). Ribonucleoprotein RNase P and protein-only RNase P (PRORP) in eukaryotes have been extensively studied, but the mechanism by which a prokaryotic nuclease recognizes and cleaves pre-tRNA is unclear. To gain insights into this mechanism, we studied homologs of Aquifex RNase P (HARPs), thought to be enzymes of approximately 23 kDa comprising only this nuclease domain. We determined the cryo-EM structure of Aq880, the first identified HARP enzyme. The structure unexpectedly revealed that Aq880 consists of both the nuclease and protruding helical (PrH) domains. Aq880 monomers assemble into a dimer via the PrH domain. Six dimers form a dodecamer with a left-handed one-turn superhelical structure. The structure also revealed that the active site of Aq880 is analogous to that of eukaryotic PRORPs. The pre-tRNA docking model demonstrated that 5' processing of pre-tRNAs is achieved by two adjacent dimers within the dodecamer. One dimer is responsible for catalysis, and the PrH domains of the other dimer are responsible for pre-tRNA elbow recognition. Our study suggests that HARPs measure an invariant distance from the pre-tRNA elbow to cleave the 5' leader sequence, which is analogous to the mechanism of eukaryotic PRORPs and the ribonucleoprotein RNase P. Collectively, these findings shed light on how different types of RNase P enzymes utilize the same pre-tRNA processing.
History
DepositionJun 16, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 11, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 23, 2022Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Assembly

Deposited unit
E: RNA-free ribonuclease P
F: RNA-free ribonuclease P
C: RNA-free ribonuclease P
D: RNA-free ribonuclease P
A: RNA-free ribonuclease P
B: RNA-free ribonuclease P
H: RNA-free ribonuclease P
G: RNA-free ribonuclease P
J: RNA-free ribonuclease P
I: RNA-free ribonuclease P
L: RNA-free ribonuclease P
K: RNA-free ribonuclease P


Theoretical massNumber of molelcules
Total (without water)273,86612
Polymers273,86612
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area40220 Å2
ΔGint-266 kcal/mol
Surface area93550 Å2

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Components

#1: Protein
RNA-free ribonuclease P / RNA-free RNase P / Protein-only RNase P


Mass: 22822.166 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus (strain VF5) (bacteria)
Strain: VF5 / Gene: aq_880 / Production host: Escherichia coli (E. coli) / References: UniProt: O67035, ribonuclease P

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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: Aq880 Dodecamer / Type: COMPLEX / Details: mono dodecamer / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.27 MDa / Experimental value: NO
Source (natural)Organism: Aquifex aeolicus (strain VF5) (bacteria) / Strain: VF5
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
150 mMTris-HCl1
2100 mMNaCl1
30.5 mMTCEP1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was mono-disperse.
Specimen supportDetails: The grid was washed by acetone prior to use. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291 K / Details: Blotting time was 5 seconds (blot force 20)

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 120000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 48.62 sec. / Electron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2370

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
4GctfCTF correction
11RELION3.1final Euler assignment
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1486899
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 238017 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00518888
ELECTRON MICROSCOPYf_angle_d0.69225428
ELECTRON MICROSCOPYf_dihedral_angle_d24.3157212
ELECTRON MICROSCOPYf_chiral_restr0.0452832
ELECTRON MICROSCOPYf_plane_restr0.0053252

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