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- EMDB-23282: polynucleotide phosphorylase -

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Basic information

Entry
Database: EMDB / ID: EMD-23282
Titlepolynucleotide phosphorylase
Map data
Sample
  • Complex: Polynucleotide phosphorylase complex in poly(A) synthesis
    • Protein or peptide: Polyribonucleotide nucleotidyltransferasePolynucleotide phosphorylase
    • RNA: poly-A RNA fragment
  • Ligand: MAGNESIUM ION
Function / homology
Function and homology information


polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mRNA catabolic process / RNA processing / magnesium ion binding / RNA binding / cytoplasm
Similarity search - Function
Guanosine pentaphosphate synthetase I/polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 / KH domain ...Guanosine pentaphosphate synthetase I/polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase / Polyribonucleotide nucleotidyltransferase, RNA-binding domain / Polyribonucleotide nucleotidyltransferase, RNA-binding domain superfamily / Polyribonucleotide nucleotidyltransferase, RNA binding domain / Exoribonuclease, phosphorolytic domain 1 / PNPase/RNase PH domain superfamily / Exoribonuclease, PH domain 2 superfamily / 3' exoribonuclease family, domain 1 / KH domain / RNA-binding domain, S1 / 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 speciesMycolicibacterium smegmatis (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsGoldgur Y / Shuman S / De La Cruz MJ / Ghosh S / Unciuleac M-C
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35-GM126945 United States
CitationJournal: RNA / Year: 2021
Title: Structure and mechanism of polynucleotide phosphorylase.
Authors: Mihaela-Carmen Unciuleac / Shreya Ghosh / M Jason de la Cruz / Yehuda Goldgur / Stewart Shuman
Abstract: Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts ...Polynucleotide phosphorylase (PNPase) catalyzes stepwise phosphorolysis of the 3'-terminal phosphodiesters of RNA chains to yield nucleoside diphosphate products. In the reverse reaction PNPase acts as a polymerase, using NDPs as substrates to add NMPs to the 3'-OH terminus of RNA chains while expelling inorganic phosphate. The apparent essentiality of PNPase for growth of militates for mycobacterial PNPase as a potential drug target. A cryo-EM structure of PNPase (MsmPNPase) reveals a characteristic ring-shaped homotrimer in which each protomer consists of two RNase PH-like domains and an intervening α-helical module on the inferior surface of the ring. The C-terminal KH and S1 domains, which impart RNA specificity to MsmPNPase, are on the opposite face of the core ring and are conformationally mobile. Single particle reconstructions of MsmPNPase in the act of poly(A) synthesis highlight a 3'-terminal (rA)4 oligonucleotide and two magnesium ions in the active site and an adenine nucleobase in the central tunnel. We identify amino acids that engage the 3' segment of the RNA chain (Phe68, Arg105, Arg112, Arg430, Arg431) and the two metal ions (Asp526, Asp532, Gln546, Asp548) and we infer those that bind inorganic phosphate (Thr470, Ser471, His435, Lys534). Alanine mutagenesis pinpointed RNA and phosphate contacts as essential (Arg105, Arg431, Lys534, Thr470+Ser471), important (Arg112, Arg430), or unimportant (Phe68) for PNPase activity. Severe phosphorolysis and polymerase defects accompanying alanine mutations of the enzymic metal ligands suggest a two-metal mechanism of catalysis by MsmPNPase.
History
DepositionJan 12, 2021-
Header (metadata) releaseJun 30, 2021-
Map releaseJun 30, 2021-
UpdateNov 30, 2022-
Current statusNov 30, 2022Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.4682712
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.4682712
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ld5
  • Surface level: 0.4682712
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23282.png

Downloads & links

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Map

FileDownload / File: emd_23282.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.064 Å
Density
Contour LevelBy AUTHOR: 0.4 / Movie #1: 0.4682712
Minimum - Maximum-4.296451 - 6.4221272
Average (Standard dev.)-0.0010494778 (±0.11011269)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 319.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0641.0641.064
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z319.200319.200319.200
α/β/γ90.00090.00090.000
start NX/NY/NZ192139186
NX/NY/NZ211274246
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-4.2966.422-0.001

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Supplemental data

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Sample components

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Entire : Polynucleotide phosphorylase complex in poly(A) synthesis

EntireName: Polynucleotide phosphorylase complex in poly(A) synthesis
Components
  • Complex: Polynucleotide phosphorylase complex in poly(A) synthesis
    • Protein or peptide: Polyribonucleotide nucleotidyltransferasePolynucleotide phosphorylase
    • RNA: poly-A RNA fragment
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Polynucleotide phosphorylase complex in poly(A) synthesis

SupramoleculeName: Polynucleotide phosphorylase complex in poly(A) synthesis
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)

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Macromolecule #1: Polyribonucleotide nucleotidyltransferase

MacromoleculeName: Polyribonucleotide nucleotidyltransferase / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO / EC number: polyribonucleotide nucleotidyltransferase
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria)
Molecular weightTheoretical: 83.647703 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGHHHHHHHH HHSSGHIEGR HMSVVELEDG VYESTAVIDN GSFGTRTIRF ETGRLAQQAA GSAVAYLDDE TMLLSATTAS KNPKDHFDF FPLTVDVEER MYAAGRIPGS FFRREGRPST DAILTCRLID RPLRPSFVDG LRNEIQVVVT VMSLDPKDLY D VLAINAAS ...String:
MGHHHHHHHH HHSSGHIEGR HMSVVELEDG VYESTAVIDN GSFGTRTIRF ETGRLAQQAA GSAVAYLDDE TMLLSATTAS KNPKDHFDF FPLTVDVEER MYAAGRIPGS FFRREGRPST DAILTCRLID RPLRPSFVDG LRNEIQVVVT VMSLDPKDLY D VLAINAAS MSTQLAGLPF SGPVGGARIA LIDGTWVAFP TVEQLERAVF DMVVAGRIVG DGDSADVAIM MVEAEATENV VE LVAGGAQ APTEAVVAEG LEAAKPFIKA LCAAQQELAD RAAKPAGEYP VFPDYEADVY DAVASVATEA LAEALTIAGK TER NDRTDE IKVEVLERLA EPYAGREKEI GAAFRSLTKK LVRQRILTDH FRIDGRGITD IRALSAEVAV IPRAHGSALF ERGE TQILG VTTLDMIKMA QQIDSLGPEN TKRYMHHYNF PPYSTGETGR VGSPKRREIG HGALAERALV PVLPSIEEFP YAIRQ VSEA LGSNGSTSMG SVCASTLALL NAGVPLKAPV AGIAMGLVSD DVDVDGKVEK RYVALTDILG AEDAFGDMDF KVAGTK DFV TALQLDTKLD GIPSQVLAGA LSQAKDARLT ILDVMAEAID RPDEMSPYAP RITTIKVPVD KIGEVIGPKG KMINSIT EE TGAQISIEDD GTVFVGAADG LSAQAAIDKI NAIANPQLPK VGERFLGTVV KTTDFGAFVS LLPGRDGLVH ISKLGKGK R IAKVEDVVKV GDKLRVEIAD IDNRGKISLV LVAEESAESA ESAGDKGAEK AEGAAADVTP AEA

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Macromolecule #2: poly-A RNA fragment

MacromoleculeName: poly-A RNA fragment / type: rna / ID: 2 / Number of copies: 3
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 2.917895 KDa
SequenceString:
AAAAAAAAA

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 6 / 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

BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL / In silico model: via cryoSPARC 3D ab-initio model
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.16.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.16.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 890249

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-7ld5:
polynucleotide phosphorylase

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