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- PDB-7og5: RNA-free Ribonuclease P from Halorhodospira halophila -

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Basic information

Entry
Database: PDB / ID: 7og5
TitleRNA-free Ribonuclease P from Halorhodospira halophila
ComponentsRNA-free ribonuclease P
KeywordsHYDROLASE / RNAseP / metallonuclease / HARP
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 speciesHalorhodospira halophila (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsAltegoer, F. / Bange, G.
CitationJournal: Elife / Year: 2021
Title: Structure and mechanistic features of the prokaryotic minimal RNase P.
Authors: Rebecca Feyh / Nadine B Waeber / Simone Prinz / Pietro Ivan Giammarinaro / Gert Bange / Georg Hochberg / Roland K Hartmann / Florian Altegoer /
Abstract: Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by ribonuclease P (RNase P) is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein- ...Endonucleolytic removal of 5'-leader sequences from tRNA precursor transcripts (pre-tRNAs) by ribonuclease P (RNase P) is essential for protein synthesis. Beyond RNA-based RNase P enzymes, protein-only versions of the enzyme exert this function in various eukarya (there termed PRORPs) and in some bacteria ( and close relatives); both enzyme types belong to distinct subgroups of the PIN domain metallonuclease superfamily. Homologs of RNase P (HARPs) are also expressed in some other bacteria and many archaea, where they coexist with RNA-based RNase P and do not represent the main RNase P activity. Here, we solved the structure of the bacterial HARP from by cryo-electron microscopy, revealing a novel screw-like dodecameric assembly. Biochemical experiments demonstrate that oligomerization is required for RNase P activity of HARPs. We propose that the tRNA substrate binds to an extended spike-helix (SH) domain that protrudes from the screw-like assembly to position the 5'-end in close proximity to the active site of the neighboring dimer. The structure suggests that eukaryotic PRORPs and prokaryotic HARPs recognize the same structural elements of pre-tRNAs (tRNA elbow region and cleavage site). Our analysis thus delivers the structural and mechanistic basis for pre-tRNA processing by the prokaryotic HARP system.
History
DepositionMay 6, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 7, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)288,61612
Polymers288,61612
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: assay for oligomerization, Mass photometry was performed to determine the dynamic oligomeric assembly
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area34590 Å2
ΔGint-170 kcal/mol
Surface area73650 Å2
MethodPISA

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Components

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


Mass: 24051.338 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Halorhodospira halophila (strain DSM 244 / SL1) (bacteria)
Strain: DSM 244 / SL1 / Gene: Hhal_2243 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A1WZ95, 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: Dodecameric assembly of minimal RNAseP system from Halorhodospira halophila
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.39 MDa / Experimental value: YES
Source (natural)Organism: Halorhodospira halophila SL1 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Details: Solutions were prepared freshly and filtered through a 0.2 um filter
Buffer component
IDConc.NameFormulaBuffer-ID
1100 mMpotassium chlorideKCl1
220 mMTris(hydroxymethyl)aminomethanTris1
SpecimenConc.: 8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: The sample was monodisperse
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K / Details: Blot for 11s with blot force -1 before plunging

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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 FIELDBright-field microscopy
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 8393

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18.2_3874refinement
PHENIX1.18.2_3874refinement
EM software
IDNameVersionCategory
7Coot0.9.3model fitting
9PHENIX1.18model refinement
13cryoSPARC3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1736597 / Symmetry type: POINT
Atomic model buildingB value: 181 / Protocol: AB INITIO MODEL / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 106.7 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.010315697
ELECTRON MICROSCOPYf_angle_d0.892721159
ELECTRON MICROSCOPYf_chiral_restr0.05072349
ELECTRON MICROSCOPYf_plane_restr0.00622775
ELECTRON MICROSCOPYf_dihedral_angle_d28.21962174

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