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- EMDB-53151: Escherichia coli polynucleotide phosphorylase in complex with rec... -

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Basic information

Entry
Database: EMDB / ID: EMD-53151
TitleEscherichia coli polynucleotide phosphorylase in complex with recognition site of RNase E
Map data
Sample
  • Complex: Polynucleotide phosphorylase in complex with recognition site from ribonuclease E
    • Protein or peptide: Polyribonucleotide nucleotidyltransferase
    • Protein or peptide: Ribonuclease E
  • Ligand: water
Keywordspolynucleotide phosphorylase / ribonuclease E / RNA degradosome / RNA BINDING PROTEIN
Function / homology
Function and homology information


regulation of RNA helicase activity / rRNA 5'-end processing / bacterial degradosome / ribonuclease E / ribonuclease E activity / polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / endoribonuclease complex / DEAD/H-box RNA helicase binding / 7S RNA binding ...regulation of RNA helicase activity / rRNA 5'-end processing / bacterial degradosome / ribonuclease E / ribonuclease E activity / polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / endoribonuclease complex / DEAD/H-box RNA helicase binding / 7S RNA binding / cyclic-di-GMP binding / RNA catabolic process / tRNA processing / mRNA catabolic process / protein complex oligomerization / RNA nuclease activity / RNA processing / RNA endonuclease activity / cytoplasmic side of plasma membrane / rRNA processing / response to heat / 3'-5'-RNA exonuclease activity / protein homotetramerization / molecular adaptor activity / tRNA binding / rRNA binding / magnesium ion binding / RNA binding / zinc ion binding / identical protein binding / membrane / cytoplasm / cytosol
Similarity search - Function
Polyribonucleotide phosphorylase C-terminal / Polyribonucleotide phosphorylase C terminal / : / 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, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase ...Polyribonucleotide phosphorylase C-terminal / Polyribonucleotide phosphorylase C terminal / : / 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, RNA-binding domain superfamily / 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 speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.52 Å
AuthorsParis G / Luisi BF
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust222451/Z/21/Z United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2025
Title: A multi-dentate, cooperative interaction between endo- and exo-ribonucleases within the bacterial RNA degradosome.
Authors: Giulia Paris / Kai Katsuya-Gaviria / Hannah Clarke / Margaret Johncock / Tom Dendooven / Aleksei Lulla / Ben F Luisi /
Abstract: In Escherichia coli and numerous other bacteria, two of the principal enzymes mediating messenger RNA decay and RNA processing-RNase E, an endoribonuclease, and polynucleotide phosphorylase (PNPase), ...In Escherichia coli and numerous other bacteria, two of the principal enzymes mediating messenger RNA decay and RNA processing-RNase E, an endoribonuclease, and polynucleotide phosphorylase (PNPase), an exoribonuclease-assemble into a multi-enzyme complex known as the RNA degradosome. While RNase E forms a homotetramer and PNPase a homotrimer, it remains unclear how these two enzymes interact within the RNA degradosome to potentially satisfy all mutual recognition sites. In this study, we used cryo-EM, biochemistry, and biophysical studies to discover and characterize a new binding mode for PNPase encompassing two or more motifs that are necessary and sufficient for strong interaction with RNase E. While a similar interaction is seen in Salmonella enterica, a different recognition mode arose for Pseudomonas aeruginosa, illustrating the evolutionary drive to maintain physical association of the two ribonucleases. The data presented here suggest a model for the quaternary organization of the RNA degradosome of E. coli, where one PNPase trimer interacts with one RNase E protomer. Conformational transitions are predicted to facilitate substrate capture and transfer to catalytic centres. The model suggests how the endo- and exo-ribonucleases might cooperate in cellular RNA turnover and recruitment of regulatory RNA by the degradosome assembly.
History
DepositionMar 14, 2025-
Header (metadata) releaseApr 23, 2025-
Map releaseApr 23, 2025-
UpdateNov 5, 2025-
Current statusNov 5, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_53151.png

Downloads & links

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Map

FileDownload / File: emd_53151.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 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: 0.062
Minimum - Maximum-0.21558419 - 0.45740148
Average (Standard dev.)0.00053260324 (±0.013129618)
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_53151_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: Second of the two half-maps

Fileemd_53151_half_map_2.map
AnnotationSecond 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: 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: Escherichia coli (E. coli)

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Macromolecule #1: Polyribonucleotide nucleotidyltransferase

MacromoleculeName: Polyribonucleotide nucleotidyltransferase / type: protein_or_peptide / ID: 1
Details: catalytic core without S1 and KH RNA binding domains
Number of copies: 3 / Enantiomer: LEVO / EC number: polyribonucleotide nucleotidyltransferase
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 59.656828 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MLNPIVRKFQ YGQHTVTLET GMMARQATAA VMVSMDDTAV FVTVVGQKKA KPGQDFFPLT VNYQERTYAA GRIPGSFFRR EGRPSEGET LIARLIDRPI RPLFPEGFVN EVQVIATVVS VNPQVNPDIV AMIGASAALS LSGIPFNGPI GAARVGYIND Q YVLNPTQD ...String:
MLNPIVRKFQ YGQHTVTLET GMMARQATAA VMVSMDDTAV FVTVVGQKKA KPGQDFFPLT VNYQERTYAA GRIPGSFFRR EGRPSEGET LIARLIDRPI RPLFPEGFVN EVQVIATVVS VNPQVNPDIV AMIGASAALS LSGIPFNGPI GAARVGYIND Q YVLNPTQD ELKESKLDLV VAGTEAAVLM VESEAQLLSE DQMLGAVVFG HEQQQVVIQN INELVKEAGK PRWDWQPEPV NE ALNARVA ALAEARLSDA YRITDKQERY AQVDVIKSET IATLLAEDET LDENELGEIL HAIEKNVVRS RVLAGEPRID GRE KDMIRG LDVRTGVLPR THGSALFTRG ETQALVTATL GTARDAQVLD ELMGERTDTF LFHYNFPPYS VGETGMVGSP KRRE IGHGR LAKRGVLAVM PDMDKFPYTV RVVSEITESN GSSSMASVCG ASLALMDAGV PIKAAVAGIA MGLVKEGDNY VVLSD ILGD EDHLGDMDFK VAGSRDGISA LQMDIKIEGI TKEIMQVALN QAKGARLHIL GVMEQAINAP RGDIS

UniProtKB: Polyribonucleotide nucleotidyltransferase

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Macromolecule #2: Ribonuclease E

MacromoleculeName: Ribonuclease E / type: protein_or_peptide / ID: 2
Details: segment of ribonuclease E that interacts with polynucleotide phosphorylase
Number of copies: 1 / Enantiomer: LEVO / EC number: ribonuclease E
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 6.14772 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
NHATAPMTRA PAPEYVPEAP RHSDWQRPTF AFEGKGAAGG HTATHHASAA PARPQPVE

UniProtKB: Ribonuclease E

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 7 / 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: 6112 / Average exposure time: 4.39 sec. / Average electron dose: 51.05 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 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: 2.52 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 108374
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-9qh0:
Escherichia coli polynucleotide phosphorylase in complex with recognition site of RNase E

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