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- PDB-8kda: Cryo-EM structure of Hydrogenobacter thermophilus minimal protein... -

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Basic information

Entry
Database: PDB / ID: 8kda
TitleCryo-EM structure of Hydrogenobacter thermophilus minimal protein-only RNase P (HARP) in complex with pre-tRNAs
Components
  • Aquifex aeolicus pre-tRNAVal
  • RNA-free ribonuclease P
KeywordsHYDROLASE/RNA / pre-tRNA processing / tRNA / pre-tRNA complex / HYDROLASE-RNA COMPLEX / RNA-free ribonuclease P / ribonuclease P
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 speciesHydrogenobacter thermophilus DSM 653 (bacteria)
Aquifex aeolicus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.19 Å
AuthorsTeramoto, T. / Adachi, N. / Yokogawa, T. / Koyasu, T. / Mayanagi, K. / 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
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)381,64122
Polymers381,52017
Non-polymers1225
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 / RNA-free RNase P / Protein-only RNase P


Mass: 21939.090 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Hydrogenobacter thermophilus DSM 653 (bacteria)
Strain: DSM 6534 / Gene: HTH_1307 / Production host: Escherichia coli (E. coli) / References: UniProt: D3DIV8, ribonuclease P
#2: RNA chain
Aquifex aeolicus pre-tRNAVal


Mass: 23650.148 Da / Num. of mol.: 5 / Source method: obtained synthetically / Source: (synth.) Aquifex aeolicus (bacteria) / References: GenBank: 6626248
#3: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY
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: Hydrogenobacter thermophilus RNA-free ribonuclease P, HARP, in complex with pre-tRNA
Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Hydrogenobacter thermophilus (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: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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 magnification: 215000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 11206

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Processing

EM software
IDNameVersionCategory
1crYOLOparticle selection
2RELION4particle selection
5GctfCTF correction
9PHENIXmodel refinement
11RELION4initial Euler assignment
12RELION4final Euler assignment
13RELION4classification
14RELION43D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1861832
3D reconstructionResolution: 3.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 191471 / Symmetry type: POINT

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