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- PDB-8cgl: Cryo-EM structure of RNase J from Helicobacter pylori -

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Basic information

Entry
Database: PDB / ID: 8cgl
TitleCryo-EM structure of RNase J from Helicobacter pylori
ComponentsRibonuclease J
KeywordsRNA BINDING PROTEIN / ribonuclease / RNase J / degradosome / Helicobacter pylori
Function / homology
Function and homology information


5'-3' RNA exonuclease activity / RNA endonuclease activity / regulation of cell growth / mRNA processing / rRNA processing / protein homotetramerization / Hydrolases; Acting on ester bonds / protein homodimerization activity / RNA binding / zinc ion binding ...5'-3' RNA exonuclease activity / RNA endonuclease activity / regulation of cell growth / mRNA processing / rRNA processing / protein homotetramerization / Hydrolases; Acting on ester bonds / protein homodimerization activity / RNA binding / zinc ion binding / identical protein binding / cytoplasm
Similarity search - Function
Ribonuclease J, conserved site / Uncharacterized protein family UPF0036 signature. / : / Ribonuclease J, bacteria / Ribonuclease J, C-terminal / Ribonuclease J C-terminal domain / Ribonuclease J, beta-CASP domain / Ribonuclease J / Ribonuclease J, domain 2 / Zn-dependent metallo-hydrolase, RNA specificity domain ...Ribonuclease J, conserved site / Uncharacterized protein family UPF0036 signature. / : / Ribonuclease J, bacteria / Ribonuclease J, C-terminal / Ribonuclease J C-terminal domain / Ribonuclease J, beta-CASP domain / Ribonuclease J / Ribonuclease J, domain 2 / Zn-dependent metallo-hydrolase, RNA specificity domain / Zn-dependent metallo-hydrolase RNA specificity domain / Metallo-beta-lactamase superfamily / Metallo-beta-lactamase superfamily / Metallo-beta-lactamase / Ribonuclease Z/Hydroxyacylglutathione hydrolase-like
Similarity search - Domain/homology
Biological speciesHelicobacter pylori B128 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsLulla, A. / Luisi, B.F.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust200873/Z/16/Z United Kingdom
CitationJournal: Nat Commun / Year: 2023
Title: Acetylation regulates the oligomerization state and activity of RNase J, the Helicobacter pylori major ribonuclease.
Authors: Alejandro Tejada-Arranz / Aleksei Lulla / Maxime Bouilloux-Lafont / Evelyne Turlin / Xue-Yuan Pei / Thibaut Douché / Mariette Matondo / Allison H Williams / Bertrand Raynal / Ben F Luisi / Hilde De Reuse /
Abstract: In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM ...In the gastric pathogen Helicobacter pylori, post-transcriptional regulation relies strongly on the activity of the essential ribonuclease RNase J. Here, we elucidated the crystal and cryo-EM structures of RNase J and determined that it assembles into dimers and tetramers in vitro. We found that RNase J extracted from H. pylori is acetylated on multiple lysine residues. Alanine substitution of several of these residues impacts on H. pylori morphology, and thus on RNase J function in vivo. Mutations of Lysine 649 modulates RNase J oligomerization in vitro, which in turn influences ribonuclease activity in vitro. Our structural analyses of RNase J reveal loops that gate access to the active site and rationalizes how acetylation state of K649 can influence activity. We propose acetylation as a regulatory level controlling the activity of RNase J and its potential cooperation with other enzymes of RNA metabolism in H. pylori.
History
DepositionFeb 5, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 27, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ribonuclease J
B: Ribonuclease J
C: Ribonuclease J
D: Ribonuclease J


Theoretical massNumber of molelcules
Total (without water)316,7884
Polymers316,7884
Non-polymers00
Water00
1
A: Ribonuclease J
D: Ribonuclease J


Theoretical massNumber of molelcules
Total (without water)158,3942
Polymers158,3942
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551
2


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
3
B: Ribonuclease J
C: Ribonuclease J


Theoretical massNumber of molelcules
Total (without water)158,3942
Polymers158,3942
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Ribonuclease J


Mass: 79196.930 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Helicobacter pylori B128 (bacteria) / Gene: rnaJ, HPB128_16g80 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C43
References: UniProt: B9XZG7, Hydrolases; Acting on ester bonds

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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: Helicobacter pylori RNase J / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.316 MDa / Experimental value: NO
Source (natural)Organism: Helicobacter pylori (bacteria) / Strain: B128
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: C43(DE3) / Plasmid: pExp-xMBP-TEV-HP_RNJ-CHis
Buffer solutionpH: 7.2
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMsodium phosphateNa2HPO41
2500 mMsodium chlorideNaCl1
30.5 mMTCEP1
SpecimenConc.: 0.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1200 nm / Calibrated defocus min: 1200 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Image recordingElectron dose: 40.58 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 34061 / Symmetry type: POINT

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