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- PDB-8j5z: The cryo-EM structure of the TwOSC1 tetramer -

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Basic information

Entry
Database: PDB / ID: 8j5z
TitleThe cryo-EM structure of the TwOSC1 tetramer
ComponentsTerpene cyclase/mutase family member
KeywordsPLANT PROTEIN / oxidosqualene cyclase / Tripterygium wilfordii Hook. f. / TwOSC1 / friedelin / cryo-EM structure
Function / homology
Function and homology information


oxidosqualene cyclase activity / triterpenoid biosynthetic process / Isomerases; Intramolecular transferases; Transferring other groups / lipid droplet
Similarity search - Function
Squalene cyclase, N-terminal / Squalene-hopene cyclase N-terminal domain / Squalene cyclase / Squalene cyclase, C-terminal / Squalene-hopene cyclase C-terminal domain / Terpenoid cyclases/protein prenyltransferase alpha-alpha toroid
Similarity search - Domain/homology
Terpene cyclase/mutase family member
Similarity search - Component
Biological speciesTripterygium wilfordii (plant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.75 Å
AuthorsMa, X. / Yuru, T. / Yunfeng, L. / Jiang, T.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Angew Chem Int Ed Engl / Year: 2023
Title: Structural and Catalytic Insight into the Unique Pentacyclic Triterpene Synthase TwOSC.
Authors: Yunfeng Luo / Xiaoli Ma / Yufan Qiu / Yun Lu / Siyu Shen / Yang Li / Haiyun Gao / Kang Chen / Jiawei Zhou / Tianyuan Hu / Lichan Tu / Huan Zhao / Dan Li / Faqiang Leng / Wei Gao / Tao Jiang ...Authors: Yunfeng Luo / Xiaoli Ma / Yufan Qiu / Yun Lu / Siyu Shen / Yang Li / Haiyun Gao / Kang Chen / Jiawei Zhou / Tianyuan Hu / Lichan Tu / Huan Zhao / Dan Li / Faqiang Leng / Wei Gao / Tao Jiang / Changli Liu / Luqi Huang / Ruibo Wu / Yuru Tong /
Abstract: The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in ...The oxidosqualene cyclase (OSC) catalyzed cyclization of the linear substrate (3S)-2,3-oxidosqualene to form diverse pentacyclic triterpenoid (PT) skeletons is one of the most complex reactions in nature. Friedelin has a unique PT skeleton involving a fascinating nine-step cation shuttle run (CSR) cascade rearrangement reaction, in which the carbocation formed at C2 moves to the other side of the skeleton, runs back to C3 to yield a friedelin cation, which is finally deprotonated. However, as crystal structure data of plant OSCs are lacking, it remains unknown why the CSR cascade reactions occur in friedelin biosynthesis, as does the exact catalytic mechanism of the CSR. In this study, we determined the first cryogenic electron microscopy structure of a plant OSC, friedelin synthase, from Tripterygium wilfordii Hook. f (TwOSC). We also performed quantum mechanics/molecular mechanics simulations to reveal the energy profile for the CSR cascade reaction and identify key residues crucial for PT skeleton formation. Furthermore, we semirationally designed two TwOSC mutants, which significantly improved the yields of friedelin and β-amyrin, respectively.
History
DepositionApr 24, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 6, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 3, 2024Group: Database references / Category: citation / citation_author
Item: _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Terpene cyclase/mutase family member
B: Terpene cyclase/mutase family member
C: Terpene cyclase/mutase family member
D: Terpene cyclase/mutase family member
hetero molecules


Theoretical massNumber of molelcules
Total (without water)354,0198
Polymers352,8504
Non-polymers1,1694
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "B"
d_3ens_1chain "C"
d_4ens_1chain "D"

NCS domain segments:

Ens-ID: ens_1

Dom-IDComponent-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11METMETGLYGLYAA1 - 7641 - 764
d_12BOGBOGBOGBOGAE801
d_21METMETGLYGLYBB1 - 7641 - 764
d_22BOGBOGBOGBOGBF801
d_31METMETGLYGLYCC1 - 7641 - 764
d_32BOGBOGBOGBOGCG801
d_41METMETGLYGLYDD1 - 7641 - 764
d_42BOGBOGBOGBOGDH801

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Components

#1: Protein
Terpene cyclase/mutase family member


Mass: 88212.500 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tripterygium wilfordii (plant) / Gene: OSC1 / Production host: Komagataella pastoris (fungus) / References: UniProt: A0A499QXI7
#2: Sugar
ChemComp-BOG / octyl beta-D-glucopyranoside / Beta-Octylglucoside / octyl beta-D-glucoside / octyl D-glucoside / octyl glucoside / Octyl glucoside


Type: D-saccharide / Mass: 292.369 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C14H28O6 / Feature type: SUBJECT OF INVESTIGATION / Comment: detergent*YM
IdentifierTypeProgram
b-octylglucosideIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
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: Tetramer of TwOSC1 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Tripterygium wilfordii (plant)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 8
Details: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM dithiothreitol, and 0.2% OG
SpecimenEmbedding 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

Software
NameVersionClassification
phenix.real_space_refine1.20.1_4487refinement
PHENIX1.20.1_4487refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 4.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 32698 / Symmetry type: POINT
RefinementCross valid method: NONE
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.001525620
ELECTRON MICROSCOPYf_angle_d0.420234792
ELECTRON MICROSCOPYf_chiral_restr0.0383580
ELECTRON MICROSCOPYf_plane_restr0.00434452
ELECTRON MICROSCOPYf_dihedral_angle_d10.48639348
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAELECTRON MICROSCOPYNCS constraints1.12099504008E-13
ens_1d_3AAELECTRON MICROSCOPYNCS constraints1.78620362039E-10
ens_1d_4AAELECTRON MICROSCOPYNCS constraints8.48378559713E-13

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