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- EMDB-32531: Cryo-EM structure of prenyltransferase domain of Macrophoma phase... -

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Basic information

Entry
Database: EMDB / ID: EMD-32531
TitleCryo-EM structure of prenyltransferase domain of Macrophoma phaseolina macrophomene synthase at 3.17 angstrom resolution
Map data
Sample
  • Organelle or cellular component: macrophomene synthase
    • Protein or peptide: macrophomene synthase
Function / homologygeranylgeranyl diphosphate synthase / Polyprenyl synthases signature 1. / isoprenoid biosynthetic process / Polyprenyl synthetase, conserved site / Polyprenyl synthetase / Polyprenyl synthetase / Isoprenoid synthase domain superfamily / Geranylgeranyl diphosphate synthase
Function and homology information
Biological speciesMacrophomina phaseolina MS6 (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.17 Å
AuthorsAdachi N / Mori T / Senda T / Abe I
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Agency for Medical Research and Development (AMED)JP20am0101071 Japan
CitationJournal: Nature / Year: 2022
Title: Discovery of non-squalene triterpenes.
Authors: Hui Tao / Lukas Lauterbach / Guangkai Bian / Rong Chen / Anwei Hou / Takahiro Mori / Shu Cheng / Ben Hu / Li Lu / Xin Mu / Min Li / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / ...Authors: Hui Tao / Lukas Lauterbach / Guangkai Bian / Rong Chen / Anwei Hou / Takahiro Mori / Shu Cheng / Ben Hu / Li Lu / Xin Mu / Min Li / Naruhiko Adachi / Masato Kawasaki / Toshio Moriya / Toshiya Senda / Xinghuan Wang / Zixin Deng / Ikuro Abe / Jeroen S Dickschat / Tiangang Liu /
Abstract: All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene. This approach is fundamentally different from the biosynthesis of short-chain (C-C) terpenes that ...All known triterpenes are generated by triterpene synthases (TrTSs) from squalene or oxidosqualene. This approach is fundamentally different from the biosynthesis of short-chain (C-C) terpenes that are formed from polyisoprenyl diphosphates. In this study, two fungal chimeric class I TrTSs, Talaromyces verruculosus talaropentaene synthase (TvTS) and Macrophomina phaseolina macrophomene synthase (MpMS), were characterized. Both enzymes use dimethylallyl diphosphate and isopentenyl diphosphate or hexaprenyl diphosphate as substrates, representing the first examples, to our knowledge, of non-squalene-dependent triterpene biosynthesis. The cyclization mechanisms of TvTS and MpMS and the absolute configurations of their products were investigated in isotopic labelling experiments. Structural analyses of the terpene cyclase domain of TvTS and full-length MpMS provide detailed insights into their catalytic mechanisms. An AlphaFold2-based screening platform was developed to mine a third TrTS, Colletotrichum gloeosporioides colleterpenol synthase (CgCS). Our findings identify a new enzymatic mechanism for the biosynthesis of triterpenes and enhance understanding of terpene biosynthesis in nature.
History
DepositionJan 3, 2022-
Header (metadata) releaseJun 22, 2022-
Map releaseJun 22, 2022-
UpdateJun 22, 2022-
Current statusJun 22, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32531.png

Downloads & links

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Map

FileDownload / File: emd_32531.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.88 Å
Density
Contour LevelBy AUTHOR: 0.079
Minimum - Maximum-0.25225127 - 0.4510748
Average (Standard dev.)-1.962123e-05 (±0.007587826)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 352.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32531_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32531_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_32531_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : macrophomene synthase

EntireName: macrophomene synthase
Components
  • Organelle or cellular component: macrophomene synthase
    • Protein or peptide: macrophomene synthase

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Supramolecule #1: macrophomene synthase

SupramoleculeName: macrophomene synthase / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all / Details: homo hexamer
Source (natural)Organism: Macrophomina phaseolina MS6 (fungus)
Molecular weightTheoretical: 480 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: macrophomene synthase

MacromoleculeName: macrophomene synthase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
SequenceString: MCNTKCYNTL AKMTVITEPA MEYMYSVPLD ESEYDKCGFC QDPRYRPRRH KDQHLARAGS AKAKELCEA LIGVYPRPTC ESAVGHSIAL VMPECMPGRV EAMGEFMESI FYMDNIAESG S QQDTGNLG TEWANDMETG PTTSVNSNTG AKQVMAKLAL QLLSIDPVCA ...String:
MCNTKCYNTL AKMTVITEPA MEYMYSVPLD ESEYDKCGFC QDPRYRPRRH KDQHLARAGS AKAKELCEA LIGVYPRPTC ESAVGHSIAL VMPECMPGRV EAMGEFMESI FYMDNIAESG S QQDTGNLG TEWANDMETG PTTSVNSNTG AKQVMAKLAL QLLSIDPVCA GNVMKAWKEW AA GFAKPRR FDSIEQYIDY RLVDSGAIVA VHLMNFGMGL DISVEELREV SDIVNHAGKA LSY QNDFFS FNYEHDMFVK LPDSIGIANA VFVLAETEGL SLAEAKERVK ELAKEHEDAV LRLK DEVES KVSYKLRICL EGLVDMVVGN LVWSASCDRY SSYRREKHQM ELPIRIQGPP TPPQE PVYE KATLPNGKQL DAPTESSGKD LSDGVATLSG DEPVLGDEIV SAPIKYLESL PSKGFR EAI IDGMNGWLNL PARSVSIIKD VVKHIHTASL LCDDIEDSSP LRRGQPSAHI IFGVSQT VN STSYLWTLAI DRLSELSSPK SLRIFIDEVR KMQIGQSFDL HWTAALQCPS EEEYLSMI D MKTGGLFHLL IRLMIAESPR KVDMDFSGLV SMTGRYFQIR DDLSNLTSEE YENQKGYCE DLDEGKYSLP LIHALKHTKN KVQLESLLIQ RKTQGGMTLE MKRLAIQIMK EAGSLEHTRK VVLELQDAV HRELAKLEEA FGQENYVIQL ALERLRIKA

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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

Concentration3.6 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Details: The grid was washed by acetone prior to use.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting time was 20 seconds (blot force 0).
DetailsThis sample was mono-disperse.

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Electron microscopy

MicroscopeTFS TALOS
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 120000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 1888 / Average exposure time: 50.24 sec. / Average electron dose: 50.0 e/Å2

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Image processing

Particle selectionNumber selected: 904572
CTF correctionSoftware - Name: Gctf
Startup modelType of model: OTHER
Details: An ab initio model was generated using RELION3's own implementation of Stochastic Gradient Descent (SGD) algorithm and low-pass filtered to 60 A for use as an initial model for 3D classification.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final 3D classificationNumber classes: 2 / Avg.num./class: 147505 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: D3 (2x3 fold dihedral) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.17 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 132926
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-7wij:
Cryo-EM structure of prenyltransferase domain of Macrophoma phaseolina macrophomene synthase

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