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- EMDB-44098: Octameric prenyltransferase core of linkerless Fusicoccadiene syn... -

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Entry
Database: EMDB / ID: EMD-44098
TitleOctameric prenyltransferase core of linkerless Fusicoccadiene synthase with two associated cyclase domains
Map dataMain map
Sample
  • Complex: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase with two associated cyclase domains
    • Protein or peptide: Fusicoccadiene synthase (linkerless)
KeywordsEnzyme / Terpene / Cyclase / Engineered Construct / TRANSFERASE
Biological speciesDiaporthe amygdali (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.79 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 14, 2024-
Header (metadata) releaseOct 9, 2024-
Map releaseOct 9, 2024-
UpdateOct 16, 2024-
Current statusOct 16, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44098.png

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Map

FileDownload / File: emd_44098.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map
Projections & slices

Image control

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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

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
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Histogram (log scale)
Contour LevelBy AUTHOR: 0.12
Minimum - Maximum-0.16013777 - 0.40817165
Average (Standard dev.)-0.00006114011 (±0.013298653)
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_44098_msk_1.map
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Additional map: Sharpened map

Fileemd_44098_additional_1.map
AnnotationSharpened map
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Half map: Half map A

Fileemd_44098_half_map_1.map
AnnotationHalf map A
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Half map: Half map B

Fileemd_44098_half_map_2.map
AnnotationHalf map B
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Sample components

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Entire : Octameric prenyltransferase core of linkerless Fusicoccadiene syn...

EntireName: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase with two associated cyclase domains
Components
  • Complex: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase with two associated cyclase domains
    • Protein or peptide: Fusicoccadiene synthase (linkerless)

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Supramolecule #1: Octameric prenyltransferase core of linkerless Fusicoccadiene syn...

SupramoleculeName: Octameric prenyltransferase core of linkerless Fusicoccadiene synthase with two associated cyclase domains
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 (linkerless)

MacromoleculeName: Fusicoccadiene synthase (linkerless) / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Diaporthe amygdali (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCGEYRGTLG PRFSFISVAV PECIPERLEV ISYANEFAFL HDDVTDHVGH DTGEVENDEM MTVFLEAAHT GAIDTSNKVD IRRAGKKRIQ SQLFLEMLAI ...String:
MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCGEYRGTLG 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 RQGLPTQHIF FEKAVLEAPY DYIASMPSKG VRDQFIDALN DWLRVPDVKV GKIKDAVRVL HNSSLLLDDF QDNSPLRRGK PSTHNIFGSA QTVNTATYSI IKAIGQIMEF SAGESVQEVM NSIMILFQGQ AMDLFWTYNG HVPSEEEYYR MIDQKTGQLF SIATSLLLNA ADNEIPRTKI QSCLHRLTRL LGRCFQIRDD YQNLVSADYT KQKGFCEDLD EGKWSLALIH MIHKQRSHMA LLNVLSTGRK HGGMTLEQKQ FVLDIIEEEK SLDYTRSVMM DLHVQLRAEI GRIEILLDSP NPAMRLLLEL LRV

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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 grids imaged: 2 / Number real images: 5558 / Average exposure time: 2.15 sec. / Average electron dose: 43.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
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: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.79 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 47363
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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