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- PDB-7xkw: The 3D strcuture of (-)-cyperene synthase with substrate analogue FSPP -
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Open data
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Basic information
Entry | Database: PDB / ID: 7xkw | ||||||||||||
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Title | The 3D strcuture of (-)-cyperene synthase with substrate analogue FSPP | ||||||||||||
![]() | (-)-cyperene synthase | ||||||||||||
![]() | PLANT PROTEIN / (-)-cyperene synthase / cyclization mechanism / directed evolution / AlphaFold2 | ||||||||||||
Function / homology | Chem-FPS![]() | ||||||||||||
Biological species | ![]() | ||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å | ||||||||||||
![]() | Yu, S.S. / Zhu, P. / Liu, Y.B. / Ma, S.G. / Ye, D. / Shao, Y.Z. / Li, W.R. / Cui, Z.J. | ||||||||||||
Funding support | ![]()
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![]() | ![]() Title: Characterization and Engineering of Two Highly Paralogous Sesquiterpene Synthases Reveal a Regioselective Reprotonation Switch. Authors: Dan Ye / Yi-Zhen Shao / Wen-Rui Li / Zhen-Jia Cui / Ting Gong / Jin-Ling Yang / Hai-Qiang Wang / Jun-Gui Dai / Ke-Ping Feng / Ming Ma / Shuang-Gang Ma / Yun-Bao Liu / Ping Zhu / Shi-Shan Yu / ![]() Abstract: Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs ...Sesquiterpene synthases (STPSs) catalyze carbocation-driven cyclization reactions that can generate structurally diverse hydrocarbons. The deprotonation-reprotonation process is widely used in STPSs to promote structural diversity, largely attributable to the distinct regio/stereoselective reprotonations. However, the molecular basis for reprotonation regioselectivity remains largely understudied. Herein, we analyzed two highly paralogous STPSs, Artabotrys hexapetalus (-)-cyperene synthase (AhCS) and ishwarane synthase (AhIS), which catalyze reactions that are distinct from the regioselective protonation of germacrene A (GA), resulting in distinct skeletons of 5/5/6 tricyclic (-)-cyperene and 6/6/5/3 tetracyclic ishwarane, respectively. Isotopic labeling experiments demonstrated that these protonations occur at C3 and C6 of GA in AhCS and AhIS, respectively. The cryo-electron microscopy-derived AhCS complex structure provided the structural basis for identifying different key active site residues that may govern their functional disparity. The structure-guided mutagenesis of these residues resulted in successful functional interconversion between AhCS and AhIS, thus targeting the three active site residues [L311-S419-C458]/[M311-V419-A458] that may act as a C3/C6 reprotonation switch for GA. These findings facilitate the rational design or directed evolution of STPSs with structurally diverse skeletons. #1: ![]() Title: Characterisation and engineering of two highly paralogous sesquiterpene synthases reveal a regioselective reprotonation switch. Authors: Ye, D. / Shao, Y.Z. / Li, W.R. / Cui, Z.J. / Gong, T. / Yang, J.L. / Wang, H.Q. / Dai, J.G. / Feng, K.P. / Ma, M. / Ma, S.G. / Liu, Y.B. / Zhu, P. / Yu, S.S. | ||||||||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 105.6 KB | Display | ![]() |
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PDB format | ![]() | 77.6 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
Others | ![]() |
-Validation report
Summary document | ![]() | 873.6 KB | Display | ![]() |
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Full document | ![]() | 879.3 KB | Display | |
Data in XML | ![]() | 21.3 KB | Display | |
Data in CIF | ![]() | 29.8 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | M: map data used to model this data |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | (-)- Mass: 65241.457 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() | ||
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#2: Chemical | ChemComp-FPS / | ||
#3: Chemical | Has ligand of interest | Y | |
-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: Complex of (-)-cyperene synthase with substrate anologue FSPP Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | |||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: ![]() | |||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | |||||||||||||||||||||||||
Buffer solution | pH: 7.5 Details: 20 uL purified AhCS protein at the concentration of 3 mg/mL was incubated with 0.3uL FSPP (10 mg/mL) on ice for 2h and then the mixture was applied to prepare cryo-EM grids. | |||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Specimen support | Grid material: GRAPHENE OXIDE / Grid type: Quantifoil R1.2/1.3 | |||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K |
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Electron microscopy imaging
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) |
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Processing
Software | Name: PHENIX / Version: 1.19.2_4158: / Classification: refinement | ||||||||||||||||||||||||
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CTF correction | Type: PHASE FLIPPING ONLY | ||||||||||||||||||||||||
3D reconstruction | Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 283635 / Symmetry type: POINT | ||||||||||||||||||||||||
Refine LS restraints |
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