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- EMDB-28220: PaFS prenyltransferase core with single interacting cyclase domai... -

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Basic information

Entry
Database: EMDB / ID: EMD-28220
TitlePaFS prenyltransferase core with single interacting cyclase domain - West class B
Map dataPaFS prenyltransferase core with single interacting cyclase domain - West class B
Sample
  • Complex: Octamer of the prenylstransferase domain of fusicoccadiene synthase from Phomopsis amygdali (PaFS)
    • Protein or peptide: Fusicoccadiene synthaseFusicocca-2,10(14)-diene synthase
Biological speciesDiaporthe amygdali (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 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
DepositionSep 23, 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_28220.png

Downloads & links

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Map

FileDownload / File: emd_28220.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPaFS prenyltransferase core with single interacting cyclase domain - West class B
Voxel sizeX=Y=Z: 1.66 Å
Density
Contour LevelBy AUTHOR: 0.0163
Minimum - Maximum-0.01524732 - 0.043464325
Average (Standard dev.)0.00018320023 (±0.0027652697)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 318.72 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_28220_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram (log scale)

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Half map: Half Map 1

Fileemd_28220_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_28220_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

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Slices (1/2)
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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: 680 KDa

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Macromolecule #1: Fusicoccadiene synthase

MacromoleculeName: Fusicoccadiene synthase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO / EC number: fusicocca-2,10(14)-diene synthase
Source (natural)Organism: Diaporthe amygdali (fungus)
SequenceString: MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCREYRGTLG PRFSFISVAV PECIPERLEV ISYANEFAFL HDDVTDHVGH DTGEVENDEM MTVFLEAAHT GAIDTSNKVD IRRAGKKRIQ SQLFLEMLAI ...String:
MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCREYRGTLG PRFSFISVAV PECIPERLEV ISYANEFAFL HDDVTDHVGH DTGEVENDEM MTVFLEAAHT GAIDTSNKVD IRRAGKKRIQ SQLFLEMLAI DPECAKTTMK SWARFVEVGS SRQHETRFVE LAKYIPYRIM DVGEMFWFGL VTFGLGLHIP DHELELCREL MANAWIAVGL QNDIWSWPKE RDAATLHGKD HVVNAIWVLM QEHQTDVDGA MQICRKLIVE YVAKYLEVIE ATKNDESISL DLRKYLDAML YSISGNVVWS LECPRYNPDV SFNKTQLEWM RQGLPSLESC PVLARSPEID SDESAVSPTA DESDSTEDSL GSGSRQDSSL STGLSLSPVH SNEGKDLQRV DTDHIFFEKA VLEAPYDYIA SMPSKGVRDQ FIDALNDWLR VPDVKVGKIK DAVRVLHNSS LLLDDFQDNS PLRRGKPSTH NIFGSAQTVN TATYSIIKAI GQIMEFSAGE SVQEVMNSIM ILFQGQAMDL FWTYNGHVPS EEEYYRMIDQ KTGQLFSIAT SLLLNAADNE IPRTKIQSCL HRLTRLLGRC FQICDDYQNL VSADYTKQKG FCEDLDEGKW SLALIHMIHK QRSHMALLNV LSTGRKHGGM TLEQKQFVLD IIEEEKSLDY TRSVMMDLHV QLRAEIGRIE ILLDSPNPAM RLLLELLRV

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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: GATAN K3 (6k 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: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 6485
FSC plot (resolution estimation)

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