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- PDB-8ih5: The cryo-EM structure of OsCyc1 that complexed with GGPP -

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Basic information

Entry
Database: PDB / ID: 8ih5
TitleThe cryo-EM structure of OsCyc1 that complexed with GGPP
ComponentsSyn-copalyl diphosphate synthase, chloroplasticSyn-copalyl-diphosphate synthase
KeywordsPLANT PROTEIN / syn-copalyl diphosphate synthase / labdane-related diterpenoid / GGPP complexed cryo-EM structure / plant defense
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
GERANYLGERANYL DIPHOSPHATE / Syn-copalyl diphosphate synthase, chloroplastic
Similarity search - Component
Biological speciesOryza sativa Japonica Group (Japanese rice)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsMa, X.L. / Xu, H.F. / 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
DepositionFeb 22, 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
F: Syn-copalyl diphosphate synthase, chloroplastic
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
hetero molecules


Theoretical massNumber of molelcules
Total (without water)533,00312
Polymers530,3016
Non-polymers2,7036
Water0
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 / Syn-copalyl-diphosphate synthase / OsCPSsyn / Syn-CPP synthase / OsCPS4 / OsCyc1


Mass: 88383.438 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
#2: Chemical
ChemComp-GRG / GERANYLGERANYL DIPHOSPHATE / Geranylgeranyl pyrophosphate


Mass: 450.443 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C20H36O7P2 / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: The D367A mutant of OsCyc1 that complexed with GGPP / Type: COMPLEX / Entity ID: #1 / 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.: 3 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 FIELDBright-field microscopy / 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

SoftwareName: PHENIX / Version: 1.17.1_3660: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 735747 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00333854
ELECTRON MICROSCOPYf_angle_d0.59445806
ELECTRON MICROSCOPYf_dihedral_angle_d18.56412574
ELECTRON MICROSCOPYf_chiral_restr0.1094976
ELECTRON MICROSCOPYf_plane_restr0.0035906

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