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- PDB-9zhj: Cryo-EM structure of RNase P RNA from Geobacillus stearothermophi... -

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Basic information

Entry
Database: PDB / ID: 9zhj
TitleCryo-EM structure of RNase P RNA from Geobacillus stearothermophilus, conformer 32
ComponentsRNase P RNA
KeywordsRNA / RNase P RNA / Geobacillus stearothermophilus / catalytic RNA / cryo-EM structure / conformational heterogeneity
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesGeobacillus stearothermophilus (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsDegenhardt, M.F.S. / Stagno, J. / Wang, Y.X.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)ZIA BC 010379, ZIC BC 011535, ZIA BC 011669 United States
CitationJournal: To Be Published
Title: Cryo-EM structure of RNase P RNA from Geobacillus stearothermophilus, conformer 32
Authors: Degenhardt, M.F.S. / Stagno, J. / Wang, Y.X.
History
DepositionDec 3, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 13, 2026Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 13, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: RNase P RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)136,54452
Polymers135,3041
Non-polymers1,24051
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNase P RNA


Mass: 135304.172 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Geobacillus stearothermophilus (bacteria) / References: GenBank: 143442
#2: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 51 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
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: ribonuclease P RNA / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.1355 MDa / Experimental value: NO
Source (natural)Organism: Geobacillus stearothermophilus (bacteria)
Source (recombinant)Organism: in vitro transcription vector pT7-TP(deltai) (others)
Buffer solutionpH: 7.5 / Details: 25 mM Tris-HCl,pH 7.5, 100 mM NaCl, 1 mM MgCl2
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris-HCl buffer1
2100 mMSodium chlorideNaCl1
31 mMMagnesium chlorideMgCl21
SpecimenConc.: 2.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: The specimen consisted of monomeric RNase P RNA from Geobacillus stearothermophilus purified and prepared in buffer containing 1 mM Magnesium. Samples were applied directly to cryo-EM grids for vitrification
Specimen supportDetails: Glow-discharged Quantifoil R1.2/1.3 Au 300 mesh grids prepared on a Vitrobot. Grids were glow-discharged on both sides for 60 s at 25 mA prior to sample application. Sample concentration was ...Details: Glow-discharged Quantifoil R1.2/1.3 Au 300 mesh grids prepared on a Vitrobot. Grids were glow-discharged on both sides for 60 s at 25 mA prior to sample application. Sample concentration was 2.7 mg/mL. A 10 s wait time and 3 s blot with force 3 to 10 at 277K were used during vitrification.
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 90 % / Chamber temperature: 277 K
Details: Grids were glow-discharged on both sides prior to vitrification. Vitrification was performed using a Vitrobot Mark IV with a 10s wait time, followed by a 3s blot and blot force 3 to 10

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Details: The alignment procedure was performed using the EPU software utilizing its autofocus and auto-tuning functions for initial microscope alignment. This was followed by manual fine-tuning ...Details: The alignment procedure was performed using the EPU software utilizing its autofocus and auto-tuning functions for initial microscope alignment. This was followed by manual fine-tuning adjustments of the beam shift, stigmation, and focus controls to ensure optimal image quality. Grid squares were screened systematically, ensuring the selection of areas with appropriate ice thickness and particle distribution. Image shift calibration and eucentric height adjustment were performed within EPU to maintain beam alignment during data acquisition
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Alignment procedure: OTHER
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 53 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 5 / Num. of real images: 78777
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2PHENIX1.21.2_5419model refinement
12cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 21475910
Details: An iterative particle-cleaning workflow was applied, combining 2D classification and heterogeneous refinement with decoy classes to achieve exhaustive particle selection.
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 85391 / Symmetry type: POINT
Atomic model buildingB value: 57 / Protocol: FLEXIBLE FIT / Space: REAL / Target criteria: maximization of map-to-model correlation
Atomic model buildingPDB-ID: 9OV3

9ov3


Pdb chain-ID: A / Accession code: 9OV3 / Chain residue range: 1-417
Details: The initial model consisted of the complete biological assembly
Pdb chain residue range: 1-417 / Source name: PDB / Type: experimental model
RefinementHighest resolution: 2.9 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210038
ELECTRON MICROSCOPYf_angle_d0.47415661
ELECTRON MICROSCOPYf_dihedral_angle_d24.5886209
ELECTRON MICROSCOPYf_chiral_restr0.0262085
ELECTRON MICROSCOPYf_plane_restr0.004417

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