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- EMDB-49478: hPNPase RNA pre-catalytic state -

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Basic information

Entry
Database: EMDB / ID: EMD-49478
TitlehPNPase RNA pre-catalytic state
Map data
Sample
  • Complex: Trimeric human PNPase bound to RNA in pre-catalytic state
    • Protein or peptide: Polyribonucleotide nucleotidyltransferase 1, mitochondrial
    • RNA: RNA (5'-R(P*AP*AP*UP*CP*UP*A)-3')
  • Ligand: SULFATE ION
  • Ligand: MAGNESIUM ION
KeywordsRNase / Protein-RNA Complex / RNA degradation / mitochondria / phosphorolytic enzyme / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homology
Function and homology information


RNA import into mitochondrion / mitochondrial mRNA polyadenylation / mitochondrial degradosome / mitochondrial mRNA catabolic process / positive regulation of mitochondrial RNA catabolic process / mitochondrial RNA 3'-end processing / Mitochondrial RNA degradation / positive regulation of miRNA catabolic process / mitochondrial RNA 5'-end processing / poly(G) binding ...RNA import into mitochondrion / mitochondrial mRNA polyadenylation / mitochondrial degradosome / mitochondrial mRNA catabolic process / positive regulation of mitochondrial RNA catabolic process / mitochondrial RNA 3'-end processing / Mitochondrial RNA degradation / positive regulation of miRNA catabolic process / mitochondrial RNA 5'-end processing / poly(G) binding / polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mitochondrial RNA catabolic process / nuclear polyadenylation-dependent mRNA catabolic process / positive regulation of mRNA catabolic process / regulation of cellular senescence / rRNA import into mitochondrion / regulation of cellular respiration / response to growth hormone / RNA catabolic process / miRNA binding / poly(U) RNA binding / protein homotrimerization / mRNA catabolic process / response to cAMP / cellular response to interferon-beta / liver regeneration / mitochondrion organization / protein homooligomerization / mitochondrial intermembrane space / mRNA processing / cellular response to oxidative stress / 3'-5'-RNA exonuclease activity / ribosome / mitochondrial matrix / endoplasmic reticulum membrane / mitochondrion / RNA binding / identical protein binding / cytosol / cytoplasm
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 1, mitochondrial
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.15 Å
AuthorsUnseld O / Das H / Hallberg BM
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Loop-mediated regulation and base flipping drive RNA cleavage by human mitochondrial PNPase.
Authors: Ole Unseld / Hrishikesh Das / B Martin Hällberg /
Abstract: Human polynucleotide phosphorylase (hPNPase), a trimeric exoribonuclease, is crucial for maintaining mitochondrial RNA metabolism, including the regulated degradation of RNA. Mutations in hPNPase ...Human polynucleotide phosphorylase (hPNPase), a trimeric exoribonuclease, is crucial for maintaining mitochondrial RNA metabolism, including the regulated degradation of RNA. Mutations in hPNPase have been linked to mitochondrial pathologies, underscoring its importance in mitochondrial RNA homeostasis. Despite this significance, the molecular basis of its catalytic mechanism and the structural consequences of active-site mutations remain poorly understood. We employed high-resolution electron cryo-microscopy to capture three distinct functional states of hPNPase during RNA degradation. In the loading state, flexible loops facilitate the recruitment of the substrate RNA and guide it toward the active site. During the pre-catalytic state, terminal nucleotides reorient within the active site, positioning the RNA backbone for cleavage, which is stabilized by Mg2+. Finally, the catalytic state reveals a nucleophilic attack of phosphate on the RNA backbone, mediated by key active-site residues. These results offer a clear biochemical framework for hPNPase-mediated RNA turnover, clarifying its catalytic mechanism and highlighting how active-site integrity is crucial for efficient RNA degradation.
History
DepositionFeb 27, 2025-
Header (metadata) releaseDec 10, 2025-
Map releaseDec 10, 2025-
UpdateDec 24, 2025-
Current statusDec 24, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49478.png

Downloads & links

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Map

FileDownload / File: emd_49478.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.)
0.85 Å/pix.
x 360 pix.
= 304.56 Å		0.85 Å/pix.
x 360 pix.
= 304.56 Å		0.85 Å/pix.
x 360 pix.
= 304.56 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.846 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0473
Minimum - Maximum-0.13501708 - 0.3061479
Average (Standard dev.)0.00007961657 (±0.007586231)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 304.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_49478_msk_1.map
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Half map: #2

Fileemd_49478_half_map_1.map
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Half map: #1

Fileemd_49478_half_map_2.map
Projections & Slices
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Sample components

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Entire : Trimeric human PNPase bound to RNA in pre-catalytic state

EntireName: Trimeric human PNPase bound to RNA in pre-catalytic state
Components
  • Complex: Trimeric human PNPase bound to RNA in pre-catalytic state
    • Protein or peptide: Polyribonucleotide nucleotidyltransferase 1, mitochondrial
    • RNA: RNA (5'-R(P*AP*AP*UP*CP*UP*A)-3')
  • Ligand: SULFATE ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Trimeric human PNPase bound to RNA in pre-catalytic state

SupramoleculeName: Trimeric human PNPase bound to RNA in pre-catalytic state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 258 KDa

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Macromolecule #1: Polyribonucleotide nucleotidyltransferase 1, mitochondrial

MacromoleculeName: Polyribonucleotide nucleotidyltransferase 1, mitochondrial
type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: polyribonucleotide nucleotidyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 84.417086 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAVAVDLGNR KLEISSGKLA RFADGSAVVQ SGDTAVMVTA VSKTKPSPSQ FMPLVVDYRQ KAAAAGRIPT NYLRREIGTS DKEILTSRI IDRSIRPLFP AGYFYDTQVL CNLLAVDGVN EPDVLAINGA SVALSLSDIP WNGPVGAVRI GIIDGEYVVN P TRKEMSSS ...String:
MAVAVDLGNR KLEISSGKLA RFADGSAVVQ SGDTAVMVTA VSKTKPSPSQ FMPLVVDYRQ KAAAAGRIPT NYLRREIGTS DKEILTSRI IDRSIRPLFP AGYFYDTQVL CNLLAVDGVN EPDVLAINGA SVALSLSDIP WNGPVGAVRI GIIDGEYVVN P TRKEMSSS TLNLVVAGAP KSQIVMLEAS AENILQQDFC HAIKVGVKYT QQIIQGIQQL VKETGVTKRT PQKLFTPSPE IV KYTHKLA MERLYAVFTD YEHDKVSRDE AVNKIRLDTE EQLKEKFPEA DPYEIIESFN VVAKEVFRSI VLNEYKRCDG RDL TSLRNV SCEVDMFKTL HGSALFQRGQ TQVLCTVTFD SLESGIKSDQ VITAINGIKD KNFMLHYEFP PYATNEIGKV TGLN RRELG HGALAEKALY PVIPRDFPFT IRVTSEVLES NGSSSMASAC GGSLALMDSG VPISSAVAGV AIGLVTKTDP EKGEI EDYR LLTDILGIED YNGDMDFKIA GTNKGITALQ ADIKLPGIPI KIVMEAIQQA SVAKKEILQI MNKTISKPRA SRKENG PVV ETVQVPLSKR AKFVGPGGYN LKKLQAETGV TISQVDEETF SVFAPTPSAM HEARDFITEI CKDDQEQQLE FGAVYTA TI TEIRDTGVMV KLYPNMTAVL LHNTQLDQRK IKHPTALGLE VGQEIQVKYF GRDPADGRMR LSRKVLQSPA TTVVRTLN D RSSIVMGEPI SQSSSNSQSG SGSAWSHPQF EKGGGSGGGS GGSAWSHPQF EK

UniProtKB: Polyribonucleotide nucleotidyltransferase 1, mitochondrial

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

MacromoleculeName: RNA (5'-R(P*AP*AP*UP*CP*UP*A)-3') / type: rna / ID: 2 / Details: RNA ordered from IDT. / Number of copies: 1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 18.64732 KDa
SequenceString:
CACAACACAC ACCACACACA CAUAAAACAA AACAGCUACG CCAUCCUCCC CCCAAUCUA

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Macromolecule #3: SULFATE ION

MacromoleculeName: SULFATE ION / type: ligand / ID: 3 / Number of copies: 3 / Formula: SO4
Molecular weightTheoretical: 96.063 Da
Chemical component information

ChemComp-SO4:
SULFATE ION

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 2 / 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

Concentration0.38 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
10.0 mMHEPESHEPES
50.0 mMNaClsodium chloride
2.0 mMMgCl2magnesium chloride
10.0 mMNa2SO4sodium sulfate
1.0 mMditiotreitolDTT
0.1 mMNaH2PO4sodium dihydrogen phosphate
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: AIR
Details: 60s back side and 120s front side glow discharching
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 4465 / Average exposure time: 2.0 sec. / Average electron dose: 48.6 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 20.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 165000
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 (ver. 4.6.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.15 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.6.2) / Number images used: 178050
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.6.2)
Final 3D classificationSoftware - Name: RELION (ver. 5.0-beta)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8-f1


Chain - Residue range: 1-783 / Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 75.34
Output model

PDB-9njb:
hPNPase RNA pre-catalytic state

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