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- EMDB-35996: The cryo-EM structure of the TwOSC1 tetramer -

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Basic information

Entry
Database: EMDB / ID: EMD-35996
TitleThe cryo-EM structure of the TwOSC1 tetramer
Map data
Sample
  • Complex: Tetramer of TwOSC1
    • Protein or peptide: Terpene cyclase/mutase family member
  • Ligand: octyl beta-D-glucopyranoside
Keywordsoxidosqualene cyclase / Tripterygium wilfordii Hook. f. / TwOSC1 / friedelin / cryo-EM structure / PLANT PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.75 Å
AuthorsMa X / Yuru T / Yunfeng L / Jiang T
Funding support1 items
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, 2023-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateJan 3, 2024-
Current statusJan 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35996.png

Downloads & links

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Map

FileDownload / File: emd_35996.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.07 Å
Density
Contour LevelBy AUTHOR: 0.0185
Minimum - Maximum-0.024605036 - 0.055030026
Average (Standard dev.)0.00025021363 (±0.0038708753)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 256.80002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_35996_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_35996_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetramer of TwOSC1

EntireName: Tetramer of TwOSC1
Components
  • Complex: Tetramer of TwOSC1
    • Protein or peptide: Terpene cyclase/mutase family member
  • Ligand: octyl beta-D-glucopyranoside

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Supramolecule #1: Tetramer of TwOSC1

SupramoleculeName: Tetramer of TwOSC1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Tripterygium wilfordii (plant)

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Macromolecule #1: Terpene cyclase/mutase family member

MacromoleculeName: Terpene cyclase/mutase family member / type: protein_or_peptide / ID: 1 / Number of copies: 4 / Enantiomer: LEVO
Source (natural)Organism: Tripterygium wilfordii (plant)
Molecular weightTheoretical: 88.2125 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: MWKLKVAERG NAPYSEYLYT TNDFSGRQTW EFDPNAGTPQ ELAKVEEARR KFTEDRHTVK PASDLLWMMQ FMREKNFKQT IPPVRLGEE EQVTYEDLTT ALTRTTNFFT ALQASDGHWP AENGGVSFFL PPFIFSLYIT GHLNSIITPE YRKEILRFIY N HQNEDGGW ...String:
MWKLKVAERG NAPYSEYLYT TNDFSGRQTW EFDPNAGTPQ ELAKVEEARR KFTEDRHTVK PASDLLWMMQ FMREKNFKQT IPPVRLGEE EQVTYEDLTT ALTRTTNFFT ALQASDGHWP AENGGVSFFL PPFIFSLYIT GHLNSIITPE YRKEILRFIY N HQNEDGGW GIHIEGHSTM FGTAFSYVCL RILGIEVDGG KDNACARARK WILDHGGITY MPSWGKTWLS ILGVYDWYGC NP MPPEFWL LPSYLPIHPA KIWCYCRMVY MPMSYLYGKR FVGPITPLIL QLREELHTQP FHEIQWRQTR HRCAKEDLYY PHS LIQDFI WDSLYVASEP LLTRWPLNKI REKALAKAME HIHYEDENSR YITIGCVEKA LCMLCCWVED PNSDYFKKHL ARIP DYLWV AEDGMKVQSF GSQLWDATFG FQALVASNLT EDEVGPALAK AYDFIKKSQV KDNPSGDFES MHRHISKGSW TFSDQ DHGW QLSDCTAEAL KCCLLAATMP QEVVGEKMKP EWVYEAINII LSLQSKSGGL AGWEPVRAGE WMEILNPMEF LENIVI EHT YVECTGSSII AFVSLKKLYP GHRTKDIDNF IRNAIRYLED VQYPDGSWYG NWGICFIYST MFALGGLAAT GRTYDNC QA VRRGVDFILK NQSDDGGWGE SYLSCPRKVY TPLDGRRSNV VQTAWAMLGL LYAGQAERDP TPLHRGAKVL INYQMEDG G YPQQEITGVF KMNCMLHYPI YRNAFPIWAL GEYRKRVPLP SKGY

UniProtKB: Terpene cyclase/mutase family member

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Macromolecule #2: octyl beta-D-glucopyranoside

MacromoleculeName: octyl beta-D-glucopyranoside / type: ligand / ID: 2 / Number of copies: 4 / Formula: BOG
Molecular weightTheoretical: 292.369 Da
Chemical component information

ChemComp-BOG:
octyl beta-D-glucopyranoside / detergent*YM / Octyl glucoside

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 8
Details: 50 mM Tris-HCl (pH 8.0), 300 mM NaCl, 2 mM dithiothreitol, and 0.2% OG
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.3000000000000003 µm / Nominal defocus min: 1.5 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.75 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 32698

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