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- EMDB-28245: PaFS prenyltransferase core with two interacting cyclase domains ... -

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Entry
Database: EMDB / ID: EMD-28245
TitlePaFS prenyltransferase core with two interacting cyclase domains - East and North
Map dataPaFS prenyltransferase core with two interacting cyclase domains - East and North
Sample
  • Complex: Octameric full-length fusicoccadiene synthase prepared with metal cofactor and inhibitor pamidronate
    • Protein or peptide: Fusicoccadiene synthase
Biological speciesDiaporthe amygdali (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.9 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 27, 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_28245.png

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Map

FileDownload / File: emd_28245.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPaFS prenyltransferase core with two interacting cyclase domains - East and North
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.66 Å/pix.
x 192 pix.
= 318.72 Å		1.66 Å/pix.
x 192 pix.
= 318.72 Å		1.66 Å/pix.
x 192 pix.
= 318.72 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.66 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.00805
Minimum - Maximum-0.017268253 - 0.046110034
Average (Standard dev.)-7.721848e-05 (±0.002733038)
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_28245_msk_1.map
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Half map: Half Map 1

Fileemd_28245_half_map_1.map
AnnotationHalf Map 1
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Half map: Half Map 2

Fileemd_28245_half_map_2.map
AnnotationHalf Map 2
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Sample components

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Entire : Octameric full-length fusicoccadiene synthase prepared with metal...

EntireName: Octameric full-length fusicoccadiene synthase prepared with metal cofactor and inhibitor pamidronate
Components
  • Complex: Octameric full-length fusicoccadiene synthase prepared with metal cofactor and inhibitor pamidronate
    • Protein or peptide: Fusicoccadiene synthase

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Supramolecule #1: Octameric full-length fusicoccadiene synthase prepared with metal...

SupramoleculeName: Octameric full-length fusicoccadiene synthase prepared with metal cofactor and inhibitor pamidronate
type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Diaporthe amygdali (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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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
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionResolution.type: BY AUTHOR / Resolution: 6.9 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 12962
Initial angle assignmentType: COMMON LINE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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