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- PDB-8i6t: The cryo-EM structure of OsCyc1 hexamer state -

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Basic information

Entry
Database: PDB / ID: 8i6t
TitleThe cryo-EM structure of OsCyc1 hexamer state
ComponentsSyn-copalyl diphosphate synthase, chloroplastic
KeywordsISOMERASE / syn-copalyl diphosphate synthase / labdane-related diterpenoids / hexamer cryo-EM structure / plant defense / PLANT PROTEIN
Function / homology
Function and homology information


syn-copalyl-diphosphate synthase / syn-copalyl diphosphate synthase activity / gibberellin biosynthetic process / terpene synthase activity / chloroplast / defense response / magnesium ion binding
Similarity search - Function
: / Terpene synthase, N-terminal domain / Terpene synthase, N-terminal domain superfamily / Terpene synthase, N-terminal domain / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid / Isoprenoid synthase domain superfamily
Similarity search - Domain/homology
Syn-copalyl diphosphate synthase, chloroplastic
Similarity search - Component
Biological speciesOryza sativa Japonica Group (Japanese rice)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.7 Å
AuthorsMa, X.L. / Xu, H.F. / Tong, Y.R. / Luo, Y.F. / Dong, Q.H. / Jiang, T.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Commun Chem / Year: 2023
Title: Structural and functional investigations of syn-copalyl diphosphate synthase from Oryza sativa.
Authors: Xiaoli Ma / Haifeng Xu / Yuru Tong / Yunfeng Luo / Qinghua Dong / Tao Jiang /
Abstract: The large superfamily of labdane-related diterpenoids is defined by the cyclization of linear geranylgeranyl pyrophosphate (GGPP), catalyzed by copalyl diphosphate synthases (CPSs) to form the basic ...The large superfamily of labdane-related diterpenoids is defined by the cyclization of linear geranylgeranyl pyrophosphate (GGPP), catalyzed by copalyl diphosphate synthases (CPSs) to form the basic decalin core, the copalyl diphosphates (CPPs). Three stereochemically distinct CPPs have been found in plants, namely (+)-CPP, ent-CPP and syn-CPP. Here, we used X-ray crystallography and cryo-EM methods to describe different oligomeric structures of a syn-copalyl diphosphate synthase from Oryza sativa (OsCyc1), and provided a cryo-EM structure of OsCyc1 mutant in complex with the substrate GGPP. Further analysis showed that tetramers are the dominant form of OsCyc1 in solution and are not necessary for enzyme activity in vitro. Through rational design, we identified an OsCyc1 mutant that can generate ent-CPP in addition to syn-CPP. Our work provides a structural and mechanistic basis for comparing different CPSs and paves the way for further enzyme design to obtain diterpene derivatives with specific chirality.
History
DepositionJan 29, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Dec 6, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Syn-copalyl diphosphate synthase, chloroplastic
B: Syn-copalyl diphosphate synthase, chloroplastic
C: Syn-copalyl diphosphate synthase, chloroplastic
D: Syn-copalyl diphosphate synthase, chloroplastic
E: Syn-copalyl diphosphate synthase, chloroplastic
F: Syn-copalyl diphosphate synthase, chloroplastic


Theoretical massNumber of molelcules
Total (without water)530,5656
Polymers530,5656
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Syn-copalyl diphosphate synthase, chloroplastic / OsCPSsyn / Syn-CPP synthase / OsCPS4 / OsCyc1


Mass: 88427.445 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Oryza sativa Japonica Group (Japanese rice)
Gene: CPS4, CYC1, Os04g0178300, LOC_Os04g09900, OSJNBa0096F01.12
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q0JF02, syn-copalyl-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: Hexamer state of OsCyc1 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Oryza sativa Japonica Group (Japanese rice)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
SpecimenConc.: 1.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2300 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: NONE
3D reconstructionResolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97919 / Symmetry type: POINT

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