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- PDB-7jth: Cryo-EM structure of unliganded octameric prenyltransferase domai... -
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Basic information
Entry | Database: PDB / ID: 7jth | ||||||
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Title | Cryo-EM structure of unliganded octameric prenyltransferase domain of Phomopsis amygdali fusicoccadiene synthase | ||||||
![]() | Fusicoccadiene synthase | ||||||
![]() | TRANSFERASE / LYASE / GGPP Synthase / Prenyltransferase | ||||||
Function / homology | ![]() fusicocca-2,10(14)-diene synthase / alcohol biosynthetic process / mycotoxin biosynthetic process / ketone biosynthetic process / geranylgeranyl diphosphate synthase / farnesyltranstransferase activity / isoprenoid biosynthetic process / lyase activity / metal ion binding Similarity search - Function | ||||||
Biological species | ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å | ||||||
![]() | Faylo, J.L. / van Eeuwen, T. / Murakami, K. / Christianson, D.W. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Structural insight on assembly-line catalysis in terpene biosynthesis. Authors: Jacque L Faylo / Trevor van Eeuwen / Hee Jong Kim / Jose J Gorbea Colón / Benjamin A Garcia / Kenji Murakami / David W Christianson / ![]() Abstract: Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a ...Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene. | ||||||
History |
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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 | ![]() | 403.3 KB | Display | ![]() |
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PDB format | ![]() | 286.2 KB | Display | ![]() |
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-Validation report
Summary document | ![]() | 1.4 MB | Display | ![]() |
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Full document | ![]() | 1.5 MB | Display | |
Data in XML | ![]() | 73.4 KB | Display | |
Data in CIF | ![]() | 111 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 22473MC 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: 83881.891 Da / Num. of mol.: 8 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() 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 |
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Sample preparation
Component | Name: Unliganded Phomopsis amygdali fusicoccadiene synthase octamer Type: COMPLEX / Details: Octamer of C-terminal prenyltransferase domain / Entity ID: all / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.392 MDa / Experimental value: NO | ||||||||||||||||||||
Source (natural) | Organism: ![]() | ||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse | ||||||||||||||||||||
Specimen support | Details: 25 mA current was applied to grid. / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: Blot for 4.5 seconds before plunging. |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN |
Image recording | Electron dose: 43 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1500 |
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Processing
Software | Name: PHENIX / Version: 1.15.2_3472: / Classification: refinement | ||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 362627 | ||||||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||||||
3D reconstruction | Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 94974 Details: Following final reconstruction, sharpening was applied using DeepEMhancer. Reconstruction before sharpening is supplied as an additional map. Symmetry type: POINT | ||||||||||||||||||||||||
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