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- EMDB-27989: Octameric prenyltransferase domain of fusicoccadiene Synthase wit... -

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Basic information

Entry
Database: EMDB / ID: EMD-27989
TitleOctameric prenyltransferase domain of fusicoccadiene Synthase with C2 symmetry sans transiently associating cyclase domains
Map dataStructure of the octameric C-terminal prenyltransferase domain of fusicoccadiene synthase from Phomopsis amygdali (PaFS) at 3.73-angstrom resolution
Sample
  • Complex: Octamer of the prenylstransferase domain of fusicoccadiene synthase from Phomopsis amygdali (PaFS)
    • Protein or peptide: Fusicoccadiene synthaseFusicocca-2,10(14)-diene synthase
Function / homology
Function and homology information


fusicocca-2,10(14)-diene synthase / alcohol biosynthetic process / mycotoxin biosynthetic process / geranylgeranyl diphosphate synthase / farnesyltranstransferase activity / isoprenoid biosynthetic process / lyase activity / metal ion binding
Similarity search - Function
Terpene synthase family 2, C-terminal metal binding / Polyprenyl synthases signature 1. / Polyprenyl synthases signature 2. / Polyprenyl synthetase, conserved site / Polyprenyl synthetase / Polyprenyl synthetase / Isoprenoid synthase domain superfamily
Similarity search - Domain/homology
Fusicoccadiene synthase
Similarity search - Component
Biological speciesDiaporthe amygdali (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.73 Å
AuthorsFaylo JL / van Eeuwen T / Christianson DW
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM56838 United States
CitationJournal: Biochemistry / Year: 2022
Title: Transient Prenyltransferase-Cyclase Association in Fusicoccadiene Synthase, an Assembly-Line Terpene Synthase.
Authors: Jacque L Faylo / Trevor van Eeuwen / Kushol Gupta / Kenji Murakami / David W Christianson /
Abstract: Fusicoccadiene synthase from the fungus (PaFS) is an assembly-line terpene synthase that catalyzes the first two steps in the biosynthesis of Fusiccocin A, a diterpene glycoside. The C-terminal ...Fusicoccadiene synthase from the fungus (PaFS) is an assembly-line terpene synthase that catalyzes the first two steps in the biosynthesis of Fusiccocin A, a diterpene glycoside. The C-terminal prenyltransferase domain of PaFS catalyzes the condensation of one molecule of C dimethylallyl diphosphate and three molecules of C isopentenyl diphosphate to form C geranylgeranyl diphosphate, which then transits to the cyclase domain for cyclization to form fusicoccadiene. Previous structural studies of PaFS using electron microscopy (EM) revealed a central octameric prenyltransferase core with eight cyclase domains tethered in random distal positions through flexible 70-residue linkers. However, proximal prenyltransferase-cyclase configurations could be captured by covalent cross-linking and observed by cryo-EM and mass spectrometry. Here, we use cryo-EM to show that proximally configured prenyltransferase-cyclase complexes are observable even in the absence of covalent cross-linking; moreover, such complexes can involve multiple cyclase domains. A conserved basic patch on the prenyltransferase domain comprises the primary touchpoint with the cyclase domain. These results support a model for transient prenyltransferase-cyclase association in which the cyclase domains of PaFS are in facile equilibrium between proximal associated and random distal positions relative to the central prenyltransferase octamer. The results of biophysical measurements using small-angle X-ray scattering, analytical ultracentrifugation, dynamic light scattering, and size-exclusion chromatography in-line with multi-angle light scattering are consistent with this model. This model accordingly provides a framework for understanding substrate transit between the prenyltransferase and cyclase domains as well as the cooperativity observed for geranylgeranyl diphosphate cyclization.
History
DepositionAug 29, 2022-
Header (metadata) releaseNov 2, 2022-
Map releaseNov 2, 2022-
UpdateNov 16, 2022-
Current statusNov 16, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27989.png

Downloads & links

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Map

FileDownload / File: emd_27989.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of the octameric C-terminal prenyltransferase domain of fusicoccadiene synthase from Phomopsis amygdali (PaFS) at 3.73-angstrom resolution
Voxel sizeX=Y=Z: 0.83 Å
Density
Contour LevelBy AUTHOR: 0.175
Minimum - Maximum-0.0018714622 - 2.0127263
Average (Standard dev.)0.0013777395 (±0.026605561)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_27989_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened map generated in non-uniform refinement of the...

Fileemd_27989_additional_1.map
AnnotationUnsharpened map generated in non-uniform refinement of the PaFS prenyltransferase octamer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1 of the octameric C-terminal prenyltransferase domain...

Fileemd_27989_half_map_1.map
AnnotationHalf-map 1 of the octameric C-terminal prenyltransferase domain of PaFS at 3.73-angstrom resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2 of the octameric C-terminal prenyltransferase domain...

Fileemd_27989_half_map_2.map
AnnotationHalf-map 2 of the octameric C-terminal prenyltransferase domain of PaFS at 3.73-angstrom resolution
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Octamer of the prenylstransferase domain of fusicoccadiene syntha...

EntireName: Octamer of the prenylstransferase domain of fusicoccadiene synthase from Phomopsis amygdali (PaFS)
Components
  • Complex: Octamer of the prenylstransferase domain of fusicoccadiene synthase from Phomopsis amygdali (PaFS)
    • Protein or peptide: Fusicoccadiene synthaseFusicocca-2,10(14)-diene synthase

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Supramolecule #1: Octamer of the prenylstransferase domain of fusicoccadiene syntha...

SupramoleculeName: Octamer of the prenylstransferase domain of fusicoccadiene synthase from Phomopsis amygdali (PaFS)
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Diaporthe amygdali (fungus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightExperimental: 392 KDa

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Macromolecule #1: Fusicoccadiene synthase

MacromoleculeName: Fusicoccadiene synthase / type: protein_or_peptide / ID: 1 / Number of copies: 8 / Enantiomer: LEVO / EC number: fusicocca-2,10(14)-diene synthase
Source (natural)Organism: Diaporthe amygdali (fungus)
Molecular weightTheoretical: 83.927977 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCREYRGTLG PRFSFISVA VPECIPERLE VISYANEFAF LHDDVTDHVG HDTGEVENDE MMTVFLEAAH TGAIDTSNKV DIRRAGKKRI Q SQLFLEML ...String:
MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCREYRGTLG PRFSFISVA VPECIPERLE VISYANEFAF LHDDVTDHVG HDTGEVENDE MMTVFLEAAH TGAIDTSNKV DIRRAGKKRI Q SQLFLEML AIDPECAKTT MKSWARFVEV GSSRQHETRF VELAKYIPYR IMDVGEMFWF GLVTFGLGLH IPDHELELCR EL MANAWIA VGLQNDIWSW PKERDAATLH GKDHVVNAIW VLMQEHQTDV DGAMQICRKL IVEYVAKYLE VIEATKNDES ISL DLRKYL DAMLYSISGN VVWSLECPRY NPDVSFNKTQ LEWMRQGLPS LESCPVLARS PEIDSDESAV SPTADESDST EDSL GSGSR QDSSLSTGLS LSPVHSNEGK DLQRVDTDHI FFEKAVLEAP YDYIASMPSK GVRDQFIDAL NDWLRVPDVK VGKIK DAVR VLHNSSLLLD DFQDNSPLRR GKPSTHNIFG SAQTVNTATY SIIKAIGQIM EFSAGESVQE VMNSIMILFQ GQAMDL FWT YNGHVPSEEE YYRMIDQKTG QLFSIATSLL LNAADNEIPR TKIQSCLHRL TRLLGRCFQI CDDYQNLVSA DYTKQKG FC EDLDEGKWSL ALIHMIHKQR SHMALLNVLS TGRKHGGMTL EQKQFVLDII EEEKSLDYTR SVMMDLHVQL RAEIGRIE I LLDSPNPAMR LLLELLRV

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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: 7.5
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: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 52.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: COMMON LINE
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.73 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 151710
FSC plot (resolution estimation)

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