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- EMDB-44155: Octameric prenyltransferase domain of linkerless Fusicoccadiene s... -

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Basic information

Entry
Database: EMDB / ID: EMD-44155
TitleOctameric prenyltransferase domain of linkerless Fusicoccadiene synthase with C2 symmetry without associated cyclase domains
Map dataMain map
Sample
  • Complex: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase
    • Protein or peptide: Fusicoccadiene synthase
KeywordsEnzyme / terpene / engineered construct / TRANSFERASE
Function / homology
Function and homology information


fusicocca-2,10(14)-diene synthase / alcohol biosynthetic process / mycotoxin biosynthetic process / geranylgeranyl diphosphate synthase / geranylgeranyl diphosphate synthase 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) / Phomopsis amygdali (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.53 Å
AuthorsWenger ES / Schultz K / Marmorstein R / 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: Proc Natl Acad Sci U S A / Year: 2024
Title: Engineering substrate channeling in a bifunctional terpene synthase.
Authors: Eliott S Wenger / Kollin Schultz / Ronen Marmorstein / David W Christianson /
Abstract: Fusicoccadiene synthase from (PaFS) is a bifunctional terpene synthase. It contains a prenyltransferase (PT) domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate ...Fusicoccadiene synthase from (PaFS) is a bifunctional terpene synthase. It contains a prenyltransferase (PT) domain that generates geranylgeranyl diphosphate (GGPP) from dimethylallyl diphosphate and three equivalents of isopentenyl diphosphate, and a cyclase domain that converts GGPP into fusicoccadiene, a precursor of the diterpene glycoside Fusicoccin A. The two catalytic domains are connected by a flexible 69-residue linker. The PT domain mediates oligomerization to form predominantly octamers, with cyclase domains randomly splayed out around the PT core. Surprisingly, despite the random positioning of cyclase domains, substrate channeling is operative in catalysis since most of the GGPP generated by the PT remains on the enzyme for cyclization. Here, we demonstrate that covalent linkage of the PT and cyclase domains is not required for GGPP channeling, although covalent linkage may improve channeling efficiency. Moreover, GGPP competition experiments with other diterpene cyclases indicate that the PaFS PT and cyclase domains are preferential partners regardless of whether they are covalently linked or not. The cryoelectron microscopy structure of the 600-kD "linkerless" construct, in which the 69-residue linker is spliced out and replaced with the tripeptide PTQ, reveals that cyclase pairs associate with all four sides of the PT octamer and exhibit fascinating quaternary structural flexibility. These results suggest that optimal substrate channeling is achieved when a cyclase domain associates with the side of the PT octamer, regardless of whether the two domains are covalently linked and regardless of whether this interaction is transient or locked in place.
History
DepositionMar 20, 2024-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44155.png

Downloads & links

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Map

FileDownload / File: emd_44155.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 384 pix.
= 414.72 Å		1.08 Å/pix.
x 384 pix.
= 414.72 Å		1.08 Å/pix.
x 384 pix.
= 414.72 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0608
Minimum - Maximum-0.11373511 - 0.24566975
Average (Standard dev.)-0.000018071993 (±0.0046030628)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 414.72003 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_44155_msk_1.map
Projections & Slices
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Additional map: DeepEMhanced map

Fileemd_44155_additional_1.map
AnnotationDeepEMhanced map
Projections & Slices
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Half map: Half map A

Fileemd_44155_half_map_1.map
AnnotationHalf map A
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_44155_half_map_2.map
AnnotationHalf map B
Projections & Slices
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Sample components

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Entire : Octameric prenyltransferase core of linkerless Fusicoccadiene synthase

EntireName: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase
Components
  • Complex: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase
    • Protein or peptide: Fusicoccadiene synthase

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Supramolecule #1: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase

SupramoleculeName: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Linkerless Fusicoccadiene synthase was generated by replacing the native 70-residue linker region of the bifunctional enzyme with the tripeptide PTQ
Source (natural)Organism: Diaporthe amygdali (fungus)
Molecular weightTheoretical: 616 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: Phomopsis amygdali (fungus)
Molecular weightTheoretical: 77.071812 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCGEYRGTLG PRFSFISVA VPECIPERLE VISYANEFAF LHDDVTDHVG HDTGEVENDE MMTVFLEAAH TGAIDTSNKV DIRRAGKKRI Q SQLFLEML ...String:
MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCGEYRGTLG 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 LEWMRQGLPT QHIFFEKAVL EAPYDYIASM PSKGVRDQFI DALN DWLRV PDVKVGKIKD AVRVLHNSSL LLDDFQDNSP LRRGKPSTHN IFGSAQTVNT ATYSIIKAIG QIMEFSAGES VQEVM NSIM ILFQGQAMDL FWTYNGHVPS EEEYYRMIDQ KTGQLFSIAT SLLLNAADNE IPRTKIQSCL HRLTRLLGRC FQIRDD YQN LVSADYTKQK GFCEDLDEGK WSLALIHMIH KQRSHMALLN VLSTGRKHGG MTLEQKQFVL DIIEEEKSLD YTRSVMM DL HVQLRAEIGR IEILLDSPNP AMRLLLELLR V

UniProtKB: Fusicoccadiene synthase

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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

Concentration1 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
50.0 mMC8H18N2O4SHEPES
200.0 mMNaClsodium chloride
1.0 mMC9H15O6PTCEP
100.0 mMC3H9NOTMAO
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 5558 / Average exposure time: 2.15 sec. / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 408639
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

8eax

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.53 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 220663
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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