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Yorodumi- PDB-8eax: Octameric prenyltransferase domain of fusicoccadiene Synthase wit... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8eax | ||||||
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Title | Octameric prenyltransferase domain of fusicoccadiene Synthase with C2 symmetry sans transiently associating cyclase domains | ||||||
Components | Fusicoccadiene synthaseFusicocca-2,10(14)-diene synthase | ||||||
Keywords | TRANSFERASE / Terpene synthase / Prenyltransferase | ||||||
Function / homology | Function and homology information fusicocca-2,10(14)-diene synthase / alcohol biosynthetic process / mycotoxin biosynthetic process / geranylgeranyl diphosphate synthase / farnesyltranstransferase activity / isoprenoid biosynthetic process / lyase activity / metal ion binding Similarity search - Function | ||||||
Biological species | Diaporthe amygdali (fungus) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.73 Å | ||||||
Authors | Faylo, J.L. / van Eeuwen, T. / Christianson, D.W. | ||||||
Funding support | United States, 1items
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Citation | Journal: Biochemistry / Year: 2022 Title: Transient Prenyltransferase-Cyclase Association in Fusicoccadiene Synthase, an Assembly-Line Terpene Synthase. Authors: Jacque L Faylo / Trevor van Eeuwen / Kushol Gupta / Kenji Murakami / David W Christianson / Abstract: Fusicoccadiene synthase from the fungus (PaFS) is an assembly-line terpene synthase that catalyzes the first two steps in the biosynthesis of Fusiccocin A, a diterpene glycoside. The C-terminal ...Fusicoccadiene synthase from the fungus (PaFS) is an assembly-line terpene synthase that catalyzes the first two steps in the biosynthesis of Fusiccocin A, a diterpene glycoside. The C-terminal prenyltransferase domain of PaFS catalyzes the condensation of one molecule of C dimethylallyl diphosphate and three molecules of C isopentenyl diphosphate to form C geranylgeranyl diphosphate, which then transits to the cyclase domain for cyclization to form fusicoccadiene. Previous structural studies of PaFS using electron microscopy (EM) revealed a central octameric prenyltransferase core with eight cyclase domains tethered in random distal positions through flexible 70-residue linkers. However, proximal prenyltransferase-cyclase configurations could be captured by covalent cross-linking and observed by cryo-EM and mass spectrometry. Here, we use cryo-EM to show that proximally configured prenyltransferase-cyclase complexes are observable even in the absence of covalent cross-linking; moreover, such complexes can involve multiple cyclase domains. A conserved basic patch on the prenyltransferase domain comprises the primary touchpoint with the cyclase domain. These results support a model for transient prenyltransferase-cyclase association in which the cyclase domains of PaFS are in facile equilibrium between proximal associated and random distal positions relative to the central prenyltransferase octamer. The results of biophysical measurements using small-angle X-ray scattering, analytical ultracentrifugation, dynamic light scattering, and size-exclusion chromatography in-line with multi-angle light scattering are consistent with this model. This model accordingly provides a framework for understanding substrate transit between the prenyltransferase and cyclase domains as well as the cooperativity observed for geranylgeranyl diphosphate cyclization. | ||||||
History |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8eax.cif.gz | 439.5 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8eax.ent.gz | 345.7 KB | Display | PDB format |
PDBx/mmJSON format | 8eax.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/ea/8eax ftp://data.pdbj.org/pub/pdb/validation_reports/ea/8eax | HTTPS FTP |
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-Related structure data
Related structure data | 27989MC 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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-Components
#1: Protein | Mass: 83927.977 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Diaporthe amygdali (fungus) / Gene: PaFS, orf1 / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: A2PZA5, fusicocca-2,10(14)-diene synthase, geranylgeranyl diphosphate synthase |
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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: Octamer of the prenylstransferase domain of fusicoccadiene synthase from Phomopsis amygdali (PaFS) Type: COMPLEX / Entity ID: all / Source: RECOMBINANT |
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Molecular weight | Value: 0.392 MDa / Experimental value: YES |
Source (natural) | Organism: Diaporthe amygdali (fungus) |
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 FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 52 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: FEI FALCON III (4k x 4k) |
-Processing
Software | Name: PHENIX / Version: dev_3736: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 151710 / Symmetry type: POINT | ||||||||||||||||||||||||
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