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- EMDB-43092: E.coli PNPase in complex with single 8-oxoG RNA -

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Basic information

Entry
Database: EMDB / ID: EMD-43092
TitleE.coli PNPase in complex with single 8-oxoG RNA
Map data
Sample
  • Complex: PNPase complexed with single 8-oxo G RNA
    • Protein or peptide: Polyribonucleotide nucleotidyltransferase
    • RNA: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*A*A*A)-3')
    • RNA: RNA (5'-R(P*AP*CP*AP*(8GM))-3')
  • Ligand: MAGNESIUM ION
KeywordsPNPase / oxidized RNA / Phosphorolysis / 8-oxo G / RNA BINDING PROTEIN
Function / homology
Function and homology information


polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mRNA catabolic process / RNA processing / 3'-5'-RNA exonuclease activity / magnesium ion binding / RNA binding / cytosol
Similarity search - Function
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 ...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
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.15 Å
AuthorsKim W / Zhang YJ
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM148356 United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2024
Title: Selective 8-oxo-rG stalling occurs in the catalytic core of polynucleotide phosphorylase (PNPase) during degradation.
Authors: Lucas G Miller / Wantae Kim / Shawn Schowe / Kathleen Taylor / Runhua Han / Vashita Jain / Raeyeon Park / Mark Sherman / Janssen Fang / Haydee Ramirez / Andrew Ellington / Phanourios Tamamis ...Authors: Lucas G Miller / Wantae Kim / Shawn Schowe / Kathleen Taylor / Runhua Han / Vashita Jain / Raeyeon Park / Mark Sherman / Janssen Fang / Haydee Ramirez / Andrew Ellington / Phanourios Tamamis / Marino J E Resendiz / Y Jessie Zhang / Lydia Contreras /
Abstract: RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) ...RNA oxidation, predominantly through the accumulation of 8-oxo-7,8-dihydroguanosine (8-oxo-rG), represents an important biomarker for cellular oxidative stress. Polynucleotide phosphorylase (PNPase) is a 3'-5' exoribonuclease that has been shown to preferentially recognize 8-oxo-rG-containing RNA and protect cells from oxidative stress. However, the impact of 8-oxo-rG on PNPase-mediated RNA degradation has not been studied. Here, we show that the presence of 8-oxo-rG in RNA leads to catalytic stalling of PNPase through in vitro RNA degradation experiments and electrophoretic analysis. We also link this stalling to the active site of the enzyme through resolution of single-particle cryo-EM structures for PNPase in complex with singly or doubly oxidized RNA oligonucleotides. Following identification of Arg399 as a key residue in recognition of both single and sequential 8-oxo-rG nucleotides, we perform follow-up in vitro analysis to confirm the importance of this residue in 8-oxo-rG-specific PNPase stalling. Finally, we investigate the effects of mutations to active site residues implicated in 8-oxo-rG binding through cell growth experiments under HO-induced oxidative stress. Specifically, Arg399 mutations show significant effects on cell growth under oxidative stress. Overall, we demonstrate that 8-oxo-rG-specific stalling of PNPase is relevant to bacterial survival under oxidative stress and speculate that this enzyme might associate with other cellular factors to mediate this stress.
History
DepositionDec 11, 2023-
Header (metadata) releaseNov 20, 2024-
Map releaseNov 20, 2024-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43092.png

Downloads & links

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Map

FileDownload / File: emd_43092.map.gz / Format: CCP4 / Size: 125 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 320 pix.
= 266.624 Å		0.83 Å/pix.
x 320 pix.
= 266.624 Å		0.83 Å/pix.
x 320 pix.
= 266.624 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8332 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0404
Minimum - Maximum-0.13574947 - 0.30406126
Average (Standard dev.)0.00036861244 (±0.010535882)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 266.624 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_43092_half_map_1.map
Projections & Slices
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Slices (1/2)
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Histogram

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Half map: #2

Fileemd_43092_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : PNPase complexed with single 8-oxo G RNA

EntireName: PNPase complexed with single 8-oxo G RNA
Components
  • Complex: PNPase complexed with single 8-oxo G RNA
    • Protein or peptide: Polyribonucleotide nucleotidyltransferase
    • RNA: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*A*A*A)-3')
    • RNA: RNA (5'-R(P*AP*CP*AP*(8GM))-3')
  • Ligand: MAGNESIUM ION

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Supramolecule #1: PNPase complexed with single 8-oxo G RNA

SupramoleculeName: PNPase complexed with single 8-oxo G RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #1: Polyribonucleotide nucleotidyltransferase

MacromoleculeName: Polyribonucleotide nucleotidyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 77.189844 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
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 RGDISEFAPR IHTIKI NPD KIKDVIGKGG SVIRALTEET GTTIEIEDDG TVKIAATDGE KAKHAIRRIE EITAEIEVGR VYTGKVTRIV DFGAFVA IG GGKEGLVHIS QIADKRVEKV TDYLQMGQEV PVKVLEVDRQ GRIRLSIKEA TEQSQPAAAP EAPAAEQGE

UniProtKB: Polyribonucleotide nucleotidyltransferase

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Macromolecule #2: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*A*A*A)-3')

MacromoleculeName: RNA (5'-R(P*AP*AP*AP*AP*AP*AP*A*A*A)-3') / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 2.917895 KDa
SequenceString:
AAAAAAAAA

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Macromolecule #3: RNA (5'-R(P*AP*CP*AP*(8GM))-3')

MacromoleculeName: RNA (5'-R(P*AP*CP*AP*(8GM))-3') / type: rna / ID: 3 / Number of copies: 3
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 1.279841 KDa
SequenceString:
ACA(8GM)

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration5 mg/mL
BufferpH: 7.4
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 69.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.9000000000000001 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 705788
Initial angle assignmentType: RANDOM ASSIGNMENT
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final 3D classificationNumber classes: 45

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