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Yorodumi- PDB-8fxi: Cryo-EM structure of Stanieria sp. CphA2 in complex with ADPCP an... -
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-Basic information
Entry | Database: PDB / ID: 8fxi | ||||||
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Title | Cryo-EM structure of Stanieria sp. CphA2 in complex with ADPCP and 4x(beta-Asp-Arg) | ||||||
Components |
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Keywords | LIGASE / cyanophycin / CphA2 / ATP-grasp | ||||||
Function / homology | Function and homology information N-acetyl-L-aspartate-L-glutamate ligase activity / ribosomal S6-glutamic acid ligase activity / SOS response / ATP binding / metal ion binding / cytoplasm Similarity search - Function | ||||||
Biological species | Stanieria sp. NIES-3757 (bacteria) synthetic construct (others) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.7 Å | ||||||
Authors | Markus, L.M. / Sharon, I. / Strauss, M. / Schmeing, T.M. | ||||||
Funding support | Canada, 1items
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Citation | Journal: Protein Sci / Year: 2023 Title: Structure and function of a hexameric cyanophycin synthetase 2. Authors: Linda M D Markus / Itai Sharon / Kim Munro / Marcel Grogg / Donald Hilvert / Mike Strauss / T Martin Schmeing / Abstract: Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store ...Cyanophycin is a natural polymer composed of a poly-aspartate backbone with arginine attached to each of the aspartate sidechains. Produced by a wide range of bacteria, which mainly use it as a store of fixed nitrogen, it has many promising industrial applications. Cyanophycin can be synthesized from the amino acids Asp and Arg by the widespread cyanophycin synthetase 1 (CphA1), or from the dipeptide β-Asp-Arg by the cyanobacterial enzyme cyanophycin synthetase 2 (CphA2). CphA2 enzymes display a range of oligomeric states, from dimers to dodecamers. Recently, the crystal structure of a CphA2 dimer was solved but could not be obtained in complex with substrate. Here, we report cryo-EM structures of the hexameric CphA2 from Stanieria sp. at ~2.8 Å resolution, both with and without ATP analog and cyanophycin. The structures show a two-fold symmetrical, trimer-of-dimers hexameric architecture, and substrate-binding interactions that are similar to those of CphA1. Mutagenesis experiments demonstrate the importance of several conserved substrate-binding residues. We also find that a Q416A/R528G double mutation prevents hexamer formation and use this double mutant to show that hexamerization augments the rate of cyanophycin synthesis. Together, these results increase our mechanistic understanding of how an interesting green polymer is biosynthesized. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8fxi.cif.gz | 722.1 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8fxi.ent.gz | 596.7 KB | Display | PDB format |
PDBx/mmJSON format | 8fxi.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8fxi_validation.pdf.gz | 1.5 MB | Display | wwPDB validaton report |
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Full document | 8fxi_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 8fxi_validation.xml.gz | 120.5 KB | Display | |
Data in CIF | 8fxi_validation.cif.gz | 178.8 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/fx/8fxi ftp://data.pdbj.org/pub/pdb/validation_reports/fx/8fxi | HTTPS FTP |
-Related structure data
Related structure data | 29534MC 8fxhC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 72886.516 Da / Num. of mol.: 6 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Stanieria sp. NIES-3757 / Gene: STA3757_02480 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A140K0M0 #2: Protein/peptide | Mass: 1103.106 Da / Num. of mol.: 2 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others) #3: Chemical | ChemComp-YHZ / | #4: Chemical | ChemComp-MG / #5: Chemical | ChemComp-ACP / | Has ligand of interest | Y | |
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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 |
-Sample preparation
Component | Name: Stanieria sp. CphA2 in complex with ADPCP and 4x(beta-Asp-Arg) Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Source (natural) | Organism: Stanieria sp. (bacteria) |
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) |
Buffer solution | pH: 7.5 |
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES |
Vitrification | Cryogen name: ETHANE |
-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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: -2.5 nm / Nominal defocus min: -1 nm |
Image recording | Electron dose: 80 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
-Processing
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION |
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3D reconstruction | Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 549663 / Symmetry type: POINT |