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- PDB-9qh3: Pseudomonas aeruginosa polynucleotide phosphorylase in complex wi... -

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Basic information

Entry
Database: PDB / ID: 9qh3
TitlePseudomonas aeruginosa polynucleotide phosphorylase in complex with recognition site of RNase E
Components
  • Polyribonucleotide nucleotidyltransferase
  • Ribonuclease E
KeywordsRNA BINDING PROTEIN / polynucleotide phosphorylase / ribonuclease E / RNA degradosome
Function / homology
Function and homology information


ribonuclease E / ribonuclease E activity / polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / RNA catabolic process / tRNA processing / mRNA catabolic process / RNA nuclease activity / RNA processing / RNA endonuclease activity ...ribonuclease E / ribonuclease E activity / polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / RNA catabolic process / tRNA processing / mRNA catabolic process / RNA nuclease activity / RNA processing / RNA endonuclease activity / cytoplasmic side of plasma membrane / rRNA processing / 3'-5'-RNA exonuclease activity / tRNA binding / rRNA binding / magnesium ion binding / RNA binding / zinc ion binding / cytosol / cytoplasm
Similarity search - Function
: / RNase E/G, Thioredoxin-like domain / Ribonuclease E/G / Ribonuclease E / RNA-binding protein AU-1/Ribonuclease E/G / Ribonuclease E/G family / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 2 ...: / RNase E/G, Thioredoxin-like domain / Ribonuclease E/G / Ribonuclease E / RNA-binding protein AU-1/Ribonuclease E/G / Ribonuclease E/G family / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 2 / 3' exoribonuclease family, domain 2 / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 / KH domain / K Homology domain, type 1 / Type-1 KH domain profile. / K Homology domain, type 1 superfamily / S1 domain profile. / Ribosomal protein S1-like RNA-binding domain / S1 RNA binding domain / S1 domain / K Homology domain / K homology RNA-binding domain / Ribosomal protein S5 domain 2-type fold / Nucleic acid-binding, OB-fold
Similarity search - Domain/homology
ADENOSINE-5'-MONOPHOSPHATE / Polyribonucleotide nucleotidyltransferase / Ribonuclease E
Similarity search - Component
Biological speciesPseudomonas aeruginosa PAO1 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsParis, G. / Luisi, B.F.
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
Wellcome Trust222451/Z/21/Z United Kingdom
CitationJournal: To Be Published
Title: Pseudomonas aeruginosa polynucleotide phosphorylase in complex with recognition site of RNase E
Authors: Paris, G. / Luisi, B.F.
History
DepositionMar 14, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 14, 2025Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 14, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Polyribonucleotide nucleotidyltransferase
B: Polyribonucleotide nucleotidyltransferase
C: Polyribonucleotide nucleotidyltransferase
D: Ribonuclease E
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,1377
Polymers182,0954
Non-polymers1,0423
Water81145
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 Polyribonucleotide nucleotidyltransferase / Polynucleotide phosphorylase / PNPase


Mass: 59727.867 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Details: catalytic core, without S1 and KH domains / Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Gene: pnp, PA4740 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: Q9HV59, polyribonucleotide nucleotidyltransferase
#2: Protein/peptide Ribonuclease E / RNase E


Mass: 2911.264 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: PNPase recognition site from RNase E / Source: (gene. exp.) Pseudomonas aeruginosa PAO1 (bacteria) / Gene: rne, PA2976 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q9HZM8, ribonuclease E
#3: Chemical ChemComp-A / ADENOSINE-5'-MONOPHOSPHATE


Type: RNA linking / Mass: 347.221 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C10H14N5O7P / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 45 / 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: Polynucleotide phosphorylase in complex with recognition site from ribonuclease E
Type: COMPLEX
Details: Complex prepared by co-expression and chromatographic purification
Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Strain: PAO1
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21(De3)
Buffer solutionpH: 8
Details: 20 mM Tris-HCl pH 8.0, 25 mM MgCl2, 150 mM KCl, 1 mM TCEP
SpecimenConc.: 3.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Purified PNPase core and RNE-muGFP-CHis proteins were mixed in 1:2 ratio and their complex were separated on the Superdex 200 Increase 10/300 GL column (Cytiva) equilibrated with Cryo-EM ...Details: Purified PNPase core and RNE-muGFP-CHis proteins were mixed in 1:2 ratio and their complex were separated on the Superdex 200 Increase 10/300 GL column (Cytiva) equilibrated with Cryo-EM buffer (20 mM Tris-HCl pH 8.0, 25 mM MgCl2, 150 mM KCl, 1 mM TCEP). Peak fractions were combined, the protein was concentrated to 15 microM using Amicon Ultra concentrator with 10 kDa cut-off (Millipore) and used to prepare Cryo-EM grids. The samples were mixed with CHAPSO (3-([3-cholamidopropyl]dimethylammonio)-2-hydroxy-1-propanesulfonate) at a final concentration of 8 mM
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277.2 K / Details: blotting force -4, 3 sec blot time

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1800 nm / Nominal defocus min: 600 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.39 sec. / Electron dose: 53.94 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4000
EM imaging opticsEnergyfilter name: TFS Selectris

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Processing

EM software
IDNameVersionCategory
1Warpparticle selection
4cryoSPARCCTF correction
7RELIONmodel fitting
9PHENIX1.20.1_4487model refinement
11cryoSPARCfinal Euler assignment
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 95237 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL / Details: phenix refine and manual rebuilding using COOT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementHighest resolution: 2.4 Å
Stereochemistry target values: REAL-SPACE (WEIGHTED MAP SUM AT ATOM CENTERS)
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412983
ELECTRON MICROSCOPYf_angle_d0.78417577
ELECTRON MICROSCOPYf_dihedral_angle_d8.1031812
ELECTRON MICROSCOPYf_chiral_restr0.0472011
ELECTRON MICROSCOPYf_plane_restr0.0382328

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