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Open data
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Basic information
Entry | Database: PDB / ID: 7ld5 | ||||||
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Title | polynucleotide phosphorylase | ||||||
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![]() | TRANSFERASE/RNA / phosphorylase polymerase RNA decay / RNA BINDING PROTEIN / TRANSFERASE-RNA complex | ||||||
Function / homology | ![]() polyribonucleotide nucleotidyltransferase / polyribonucleotide nucleotidyltransferase activity / mRNA catabolic process / RNA processing / magnesium ion binding / RNA binding / cytoplasm Similarity search - Function | ||||||
Biological species | ![]() synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.07 Å | ||||||
![]() | Goldgur, Y. / Shuman, S. / De La Cruz, M.J. / Ghosh, S. / Unciuleac, M.-C. | ||||||
Funding support | ![]()
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![]() | ![]() 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. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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PDBx/mmCIF format | ![]() | 307.2 KB | Display | ![]() |
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PDB format | ![]() | 245.7 KB | Display | ![]() |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 23282MC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 83647.703 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() References: UniProt: A0QVQ5, polyribonucleotide nucleotidyltransferase #2: RNA chain | Mass: 2917.895 Da / Num. of mol.: 3 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #3: Chemical | ChemComp-MG / Has ligand of interest | N | Sequence details | Authors state that the exact length of RNA fragment is unknown. | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Polynucleotide phosphorylase complex in poly(A) synthesis Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES | ||||||||||||
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Source (natural) |
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Source (recombinant) | Organism: ![]() ![]() | ||||||||||||
Buffer solution | pH: 8 | ||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||
Vitrification | Cryogen name: ETHANE |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 53 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.17.1_3660: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 890249 / Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | Space: REAL | ||||||||||||||||||||||||
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