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- EMDB-12878: RNA-free Ribonuclease P from Halorhodospira halophila -

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Basic information

Entry
Database: EMDB / ID: EMD-12878
TitleRNA-free Ribonuclease P from Halorhodospira halophila
Map data
Sample
  • Complex: Dodecameric assembly of minimal RNAseP system from Halorhodospira halophila
    • Protein or peptide: RNA-free ribonuclease P
KeywordsRNAseP / metallonuclease / HARP / HYDROLASE
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 SL1 (bacteria) / Halorhodospira halophila (strain DSM 244 / SL1) (bacteria)
Methodsingle 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, 2021-
Header (metadata) releaseJul 7, 2021-
Map releaseJul 7, 2021-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.124
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.124
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7og5
  • Surface level: 0.124
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12878.png

Downloads & links

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Map

FileDownload / File: emd_12878.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 256 pix.
= 213.248 Å		0.83 Å/pix.
x 256 pix.
= 213.248 Å		0.83 Å/pix.
x 256 pix.
= 213.248 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.833 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.124 / Movie #1: 0.124
Minimum - Maximum-0.62514126 - 0.99049455
Average (Standard dev.)0.0019421679 (±0.031905625)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 213.248 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8330.8330.833
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z213.248213.248213.248
α/β/γ90.00090.00090.000
start NX/NY/NZ192139186
NX/NY/NZ211274246
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.6250.9900.002

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Supplemental data

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Sample components

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Entire : Dodecameric assembly of minimal RNAseP system from Halorhodospira...

EntireName: Dodecameric assembly of minimal RNAseP system from Halorhodospira halophila
Components
  • Complex: Dodecameric assembly of minimal RNAseP system from Halorhodospira halophila
    • Protein or peptide: RNA-free ribonuclease P

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Supramolecule #1: Dodecameric assembly of minimal RNAseP system from Halorhodospira...

SupramoleculeName: Dodecameric assembly of minimal RNAseP system from Halorhodospira halophila
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Halorhodospira halophila SL1 (bacteria)
Molecular weightTheoretical: 390 KDa

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Macromolecule #1: RNA-free ribonuclease P

MacromoleculeName: RNA-free ribonuclease P / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO / EC number: ribonuclease P
Source (natural)Organism: Halorhodospira halophila (strain DSM 244 / SL1) (bacteria)
Strain: DSM 244 / SL1
Molecular weightTheoretical: 24.051338 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: GSHMASRRFV LDTSVFTNPD VYLRFDEEPM QAISVFLGLA RRADAEFYMP GPVYQELCNL RSMDLIGAEF ETEVYIRSPR RFSMTIPSE VLYEFIEEVR TRIQRGLRIA EEHARQAGQA ESLPPELITQ LRERYREAMR RGILDSREDI DVVLLAYELD A TLVSADEG ...String:
GSHMASRRFV LDTSVFTNPD VYLRFDEEPM QAISVFLGLA RRADAEFYMP GPVYQELCNL RSMDLIGAEF ETEVYIRSPR RFSMTIPSE VLYEFIEEVR TRIQRGLRIA EEHARQAGQA ESLPPELITQ LRERYREAMR RGILDSREDI DVVLLAYELD A TLVSADEG MRKFAERIGI KLVNPRYLRG VMQNLAGDDP GHAPPCGPDQ PAG

UniProtKB: RNA-free ribonuclease P

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration8 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
100.0 mMKClpotassium chloride
20.0 mMTrisTris(hydroxymethyl)aminomethan

Details: Solutions were prepared freshly and filtered through a 0.2 um filter
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 11s with blot force -1 before plunging.
DetailsThe sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 8393 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.1) / Number images used: 1736597
Initial angle assignmentType: PROJECTION MATCHING
Final angle assignmentType: PROJECTION MATCHING
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 181
Output model

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

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