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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
TitleCryo-EM structure of unliganded octameric prenyltransferase domain of Phomopsis amygdali fusicoccadiene synthase
ComponentsFusicoccadiene synthase
KeywordsTRANSFERASE / LYASE / GGPP Synthase / Prenyltransferase
Function / homology
Function and homology information


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
Terpene synthase family 2, C-terminal metal binding / Polyprenyl synthases signature 1. / Polyprenyl synthases signature 2. / Polyprenyl synthetase, conserved site / Polyprenyl synthetase / Polyprenyl synthetase / Isoprenoid synthase domain superfamily
Similarity search - Domain/homology
Fusicoccadiene synthase
Similarity search - Component
Biological speciesPhomopsis amygdali (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsFaylo, J.L. / van Eeuwen, T. / Murakami, K. / Christianson, D.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM56838 United States
CitationJournal: Nat Commun / Year: 2021
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
DepositionAug 17, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 28, 2021Provider: repository / Type: Initial release
Revision 1.1Jul 28, 2021Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2May 29, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: Fusicoccadiene synthase
B: Fusicoccadiene synthase
C: Fusicoccadiene synthase
D: Fusicoccadiene synthase
E: Fusicoccadiene synthase
F: Fusicoccadiene synthase
G: Fusicoccadiene synthase
H: Fusicoccadiene synthase


Theoretical massNumber of molelcules
Total (without water)671,0558
Polymers671,0558
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: cross-linking, gel filtration, light scattering, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Fusicoccadiene synthase / FS / Fusicoccin A biosynthetic gene clusters protein 1 / PaDC4:GGS


Mass: 83881.891 Da / Num. of mol.: 8
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Phomopsis amygdali (fungus) / Gene: PaFS, orf1 / Plasmid: pET28a(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21-CodonPlus (DE3)-RIL
References: UniProt: A2PZA5, fusicocca-2,10(14)-diene synthase, geranylgeranyl diphosphate synthase

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Unliganded Phomopsis amygdali fusicoccadiene synthase octamer
Type: COMPLEX / Details: Octamer of C-terminal prenyltransferase domain / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.392 MDa / Experimental value: NO
Source (natural)Organism: Phomopsis amygdali (fungus)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: BL21-CodonPlus (DE3)-RIL / Plasmid: pET28a(+)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid1
2150 mMSodium chlorideNaCl1
31.5 mMtris(2-carboxyethyl)phosphine1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
Specimen supportDetails: 25 mA current was applied to grid. / Grid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: 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
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron 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

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
13cryoSPARC2.5.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 362627
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 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
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00317736
ELECTRON MICROSCOPYf_angle_d0.57424020
ELECTRON MICROSCOPYf_dihedral_angle_d8.60412656
ELECTRON MICROSCOPYf_chiral_restr0.0372812
ELECTRON MICROSCOPYf_plane_restr0.0053052

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