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- EMDB-53153: Pseudomonas aeruginosa polynucleotide phosphorylase in complex wi... -

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Basic information

Entry
Database: EMDB / ID: EMD-53153
TitlePseudomonas aeruginosa polynucleotide phosphorylase in complex with recognition site of RNase E
Map dataSharpened map
Sample
  • Complex: Polynucleotide phosphorylase in complex with recognition site from ribonuclease E
    • Protein or peptide: Polyribonucleotide nucleotidyltransferase
    • Protein or peptide: Ribonuclease E
  • Ligand: ADENOSINE-5'-MONOPHOSPHATE
  • Ligand: water
Keywordspolynucleotide phosphorylase / ribonuclease E / RNA degradosome / RNA BINDING PROTEIN
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
Polyribonucleotide nucleotidyltransferase / Ribonuclease E
Similarity search - Component
Biological speciesPseudomonas aeruginosa (bacteria) / Pseudomonas aeruginosa PAO1 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.4 Å
AuthorsParis G / Luisi BF
Funding support United Kingdom, 1 items
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 BF
History
DepositionMar 14, 2025-
Header (metadata) releaseMay 14, 2025-
Map releaseMay 14, 2025-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53153.png

Downloads & links

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Map

FileDownload / File: emd_53153.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 360 pix.
= 262.8 Å		0.73 Å/pix.
x 360 pix.
= 262.8 Å		0.73 Å/pix.
x 360 pix.
= 262.8 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.73 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.058
Minimum - Maximum-0.23313108 - 0.47963125
Average (Standard dev.)0.000396145 (±0.015105055)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 262.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: One of the two half-maps

Fileemd_53153_half_map_1.map
AnnotationOne of the two half-maps
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: The second of the two half-maps

Fileemd_53153_half_map_2.map
AnnotationThe second of the two half-maps
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Polynucleotide phosphorylase in complex with recognition site fro...

EntireName: Polynucleotide phosphorylase in complex with recognition site from ribonuclease E
Components
  • Complex: Polynucleotide phosphorylase in complex with recognition site from ribonuclease E
    • Protein or peptide: Polyribonucleotide nucleotidyltransferase
    • Protein or peptide: Ribonuclease E
  • Ligand: ADENOSINE-5'-MONOPHOSPHATE
  • Ligand: water

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Supramolecule #1: Polynucleotide phosphorylase in complex with recognition site fro...

SupramoleculeName: Polynucleotide phosphorylase in complex with recognition site from ribonuclease E
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Details: Complex prepared by co-expression and chromatographic purification
Source (natural)Organism: Pseudomonas aeruginosa (bacteria) / Strain: PAO1

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Macromolecule #1: Polyribonucleotide nucleotidyltransferase

MacromoleculeName: Polyribonucleotide nucleotidyltransferase / type: protein_or_peptide / ID: 1 / Details: catalytic core, without S1 and KH domains / Number of copies: 3 / Enantiomer: LEVO / EC number: polyribonucleotide nucleotidyltransferase
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 59.727867 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MNPVTKQFQF GQSTVTLETG RIARQATGAV LVTMDDVSVL VTVVGAKSPA EGRDFFPLSV HYQEKTYAAG RIPGGFFKRE GRPSEKETL TSRLIDRPIR PLFPEGFMNE VQVVCTVVST NKKSDPDIAA MIGTSAALAI SGIPFAGPIG AARVGFHPEI G YILNPTYE ...String:
MNPVTKQFQF GQSTVTLETG RIARQATGAV LVTMDDVSVL VTVVGAKSPA EGRDFFPLSV HYQEKTYAAG RIPGGFFKRE GRPSEKETL TSRLIDRPIR PLFPEGFMNE VQVVCTVVST NKKSDPDIAA MIGTSAALAI SGIPFAGPIG AARVGFHPEI G YILNPTYE QLQSSSLDMV VAGTEDAVLM VESEADELTE DQMLGAVLFA HDEFQAVIRA VKELAAEAGK PAWDWKAPAE NT VLVNAIK AELGEAISQA YTITIKQDRY NRLGELRDQA VALFAGEEEG KFPASEVKDV FGLLEYRTVR ENIVNGKPRI DGR DTRTVR PLRIEVGVLG KTHGSALFTR GETQALVVAT LGTARDAQLL DTLEGERKDA FMLHYNFPPF SVGECGRMGS PGRR EIGHG RLARRGVAAM LPTQDEFPYT IRVVSEITES NGSSSMASVC GASLALMDAG VPVKAPVAGI AMGLVKEGEK FAVLT DILG DEDHLGDMDF KVAGTDKGVT ALQMDIKING ITEEIMEIAL GQALEARLNI LGQMNQVIAK PRAELSENAP

UniProtKB: Polyribonucleotide nucleotidyltransferase

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Macromolecule #2: Ribonuclease E

MacromoleculeName: Ribonuclease E / type: protein_or_peptide / ID: 2 / Details: PNPase recognition site from RNase E / Number of copies: 1 / Enantiomer: LEVO / EC number: ribonuclease E
Source (natural)Organism: Pseudomonas aeruginosa PAO1 (bacteria)
Molecular weightTheoretical: 2.911264 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
VPANATGRAL NDPREKRRLQ REAER

UniProtKB: Ribonuclease E

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Macromolecule #3: ADENOSINE-5'-MONOPHOSPHATE

MacromoleculeName: ADENOSINE-5'-MONOPHOSPHATE / type: ligand / ID: 3 / Number of copies: 3 / Formula: A
Molecular weightTheoretical: 347.221 Da
Chemical component information

ChemComp-A:
ADENOSINE-5'-MONOPHOSPHATE

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 45 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration3.6 mg/mL
BufferpH: 8
Details: 20 mM Tris-HCl pH 8.0, 25 mM MgCl2, 150 mM KCl, 1 mM TCEP
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.2 K / Instrument: FEI VITROBOT MARK IV / Details: blotting force -4, 3 sec blot time.
DetailsPurified 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

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number grids imaged: 1 / Number real images: 4000 / Average exposure time: 4.39 sec. / Average electron dose: 53.94 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.6 µm
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

CTF correctionSoftware - Name: cryoSPARC / Type: NONE
Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 95237
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Detailsphenix refine and manual rebuilding using COOT
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-9qh3:
Pseudomonas aeruginosa polynucleotide phosphorylase in complex with recognition site of RNase E

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