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- PDB-8kd9: Cryo-EM structure of Aquifex aeolicus minimal protein-only RNase ... -

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Basic information

Entry
Database: PDB / ID: 8kd9
TitleCryo-EM structure of Aquifex aeolicus minimal protein-only RNase P (HARP) in complex with pre-tRNAs
Components
  • Aquifex aeolicus pre-tRNAVal
  • RNA-free ribonuclease P
KeywordsHYDROLASE-RNA COMPLEX / pre-tRNA processing / tRNA / pre-tRNA complex / RNA-free ribonuclease P / ribonuclease
Function / homology
Function and homology information


ribonuclease P / ribonuclease P activity / tRNA 5'-leader removal
Similarity search - Function
RNA-free ribonuclease P / PINc domain ribonuclease / PIN-like domain superfamily
Similarity search - Domain/homology
: / RNA / RNA (> 10) / RNA-free ribonuclease P
Similarity search - Component
Biological speciesAquifex aeolicus VF5 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.87 Å
AuthorsTeramoto, T. / Koyasu, T. / Mayanagi, K. / Yokogawa, T. / Adachi, N. / Nakamura, T. / Senda, T. / Kakuta, Y.
Funding support Japan, 3items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS)21K06032 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101071 Japan
Japan Agency for Medical Research and Development (AMED)JP21am0101091 Japan
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis of transfer RNA processing by bacterial minimal RNase P.
Authors: Takamasa Teramoto / Takeshi Koyasu / Takashi Yokogawa / Naruhiko Adachi / Kouta Mayanagi / Takahiro Nakamura / Toshiya Senda / Yoshimitsu Kakuta /
Abstract: Precursor tRNAs (pre-tRNAs) require nucleolytic removal of 5'-leader and 3'-trailer sequences for maturation, which is essential for proper tRNA function. The endoribonuclease RNase P exists in ...Precursor tRNAs (pre-tRNAs) require nucleolytic removal of 5'-leader and 3'-trailer sequences for maturation, which is essential for proper tRNA function. The endoribonuclease RNase P exists in diverse forms, including RNA- and protein-based RNase P, and removes 5'-leader sequences from pre-tRNAs. Some bacteria and archaea possess a unique minimal protein-based RNase P enzyme, HARP, which forms dodecamers with twelve active sites. Here, we present cryogenic electron microscopy structures of HARP dodecamers complexed with five pre-tRNAs, and we show that HARP oligomerization enables specific recognition of the invariant distance between the acceptor stem 5'-end and the TψC-loop, functioning as a molecular ruler-a feature representing convergent evolution among RNase P enzymes. The HARP dodecamer uses only five active sites for 5'-leader cleavage, while we identify a 3'-trailer cleavage activity in the remaining seven sites. This elucidation reveals how small proteins evolve through oligomerization to adapt a pivotal biological function (5'-leader processing) and acquire a novel function (3'-trailer processing).
History
DepositionAug 9, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 14, 2024Provider: repository / Type: Initial release
Revision 1.0Aug 14, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Aug 14, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.1Jun 25, 2025Group: Data collection / Structure summary / Category: em_admin / em_software / pdbx_entry_details / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jun 25, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name
Revision 1.2Jul 9, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA-free ribonuclease P
B: RNA-free ribonuclease P
C: RNA-free ribonuclease P
D: RNA-free ribonuclease P
E: RNA-free ribonuclease P
F: RNA-free ribonuclease P
G: RNA-free ribonuclease P
H: RNA-free ribonuclease P
I: RNA-free ribonuclease P
J: RNA-free ribonuclease P
K: RNA-free ribonuclease P
L: RNA-free ribonuclease P
T: Aquifex aeolicus pre-tRNAVal
M: Aquifex aeolicus pre-tRNAVal
N: Aquifex aeolicus pre-tRNAVal
O: Aquifex aeolicus pre-tRNAVal
P: Aquifex aeolicus pre-tRNAVal


Theoretical massNumber of molelcules
Total (without water)385,16017
Polymers385,16017
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
RNA-free ribonuclease P


Mass: 22242.473 Da / Num. of mol.: 12 / Mutation: C114S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Aquifex aeolicus VF5 (bacteria) / Gene: aq_880 / Production host: Escherichia coli (E. coli) / References: UniProt: O67035
#2: RNA chain
Aquifex aeolicus pre-tRNAVal


Mass: 23650.148 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) Aquifex aeolicus VF5 (bacteria) / References: GenBank: 6626248
Has protein modificationN

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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: Aquifex aeolicus RNA-free ribonuclease P, HARP, in complex with pre-tRNA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Aquifex aeolicus (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
125 mMTris-HCl1
2100 mMNaCl1
31 mMTCEP1
45 mMMgCl21
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

MicroscopyModel: JEOL CRYO ARM 300
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 60000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 55 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 8362

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Processing

EM software
IDNameVersionCategory
1RELION3.1.3particle selection
4CTFFIND4.1CTF correction
8PHENIXmodel refinement
10RELION3.1.3initial Euler assignment
11RELION3.1.3final Euler assignment
12RELION3.1.3classification
13RELION3.1.33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4522335
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.87 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 578312 / Symmetry type: POINT

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