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- EMDB-22473: Cryo-EM structure of unliganded octameric prenyltransferase domai... -

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Basic information

Entry
Database: EMDB / ID: EMD-22473
TitleCryo-EM structure of unliganded octameric prenyltransferase domain of Phomopsis amygdali fusicoccadiene synthase
Map dataSharpened map of PaFS C-terminal prenyltransferase octamer with C2 symmetry
Sample
  • Complex: Unliganded Phomopsis amygdali fusicoccadiene synthase octamer
    • 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 speciesPhomopsis amygdali (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsFaylo JL / van Eeuwen T / Murakami K / 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: Nat Commun / Year: 2021
Title: Structural insight on assembly-line catalysis in terpene biosynthesis.
Authors: Jacque L Faylo / Trevor van Eeuwen / Hee Jong Kim / Jose J Gorbea Colón / Benjamin A Garcia / Kenji Murakami / David W Christianson /
Abstract: Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a ...Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is a unique bifunctional terpenoid synthase that catalyzes the first two steps in the biosynthesis of the diterpene glycoside Fusicoccin A, a mediator of 14-3-3 protein interactions. The prenyltransferase domain of PaFS generates geranylgeranyl diphosphate, which the cyclase domain then utilizes to generate fusicoccadiene, the tricyclic hydrocarbon skeleton of Fusicoccin A. Here, we use cryo-electron microscopy to show that the structure of full-length PaFS consists of a central octameric core of prenyltransferase domains, with the eight cyclase domains radiating outward via flexible linker segments in variable splayed-out positions. Cryo-electron microscopy and chemical crosslinking experiments additionally show that compact conformations can be achieved in which cyclase domains are more closely associated with the prenyltransferase core. This structural analysis provides a framework for understanding substrate channeling, since most of the geranylgeranyl diphosphate generated by the prenyltransferase domains remains on the enzyme for cyclization to form fusicoccadiene.
History
DepositionAug 17, 2020-
Header (metadata) releaseApr 28, 2021-
Map releaseApr 28, 2021-
UpdateJul 28, 2021-
Current statusJul 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.141
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.141
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jth
  • Surface level: 0.141
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22473.png

Downloads & links

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Map

FileDownload / File: emd_22473.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map of PaFS C-terminal prenyltransferase octamer with C2 symmetry
Voxel sizeX=Y=Z: 1.142 Å
Density
Contour LevelBy AUTHOR: 0.141 / Movie #1: 0.141
Minimum - Maximum-0.0017603644 - 1.8553731
Average (Standard dev.)0.00060603966 (±0.019564489)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 438.52798 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.1421.1421.142
M x/y/z384384384
origin x/y/z0.0000.0000.000
length x/y/z438.528438.528438.528
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS384384384
D min/max/mean-0.0021.8550.001

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Supplemental data

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Mask #1

Fileemd_22473_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened, unfiltered reconstruction of PaFS C-terminal prenyltransferase octamer...

Fileemd_22473_additional_1.map
AnnotationUnsharpened, unfiltered reconstruction of PaFS C-terminal prenyltransferase octamer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened, unfiltered half-map of PaFS C-terminal prenyltransferase octamer...

Fileemd_22473_half_map_1.map
AnnotationUnsharpened, unfiltered half-map of PaFS C-terminal prenyltransferase octamer with C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Unsharpened, unfiltered half-map of PaFS C-terminal prenyltransferase octamer...

Fileemd_22473_half_map_2.map
AnnotationUnsharpened, unfiltered half-map of PaFS C-terminal prenyltransferase octamer with C2 symmetry
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Unliganded Phomopsis amygdali fusicoccadiene synthase octamer

EntireName: Unliganded Phomopsis amygdali fusicoccadiene synthase octamer
Components
  • Complex: Unliganded Phomopsis amygdali fusicoccadiene synthase octamer
    • Protein or peptide: Fusicoccadiene synthaseFusicocca-2,10(14)-diene synthase

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Supramolecule #1: Unliganded Phomopsis amygdali fusicoccadiene synthase octamer

SupramoleculeName: Unliganded Phomopsis amygdali fusicoccadiene synthase octamer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Octamer of C-terminal prenyltransferase domain
Source (natural)Organism: Phomopsis amygdali (fungus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21-CodonPlus (DE3)-RIL / Recombinant plasmid: pET28a(+)
Molecular weightTheoretical: 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: Phomopsis amygdali (fungus)
Molecular weightTheoretical: 83.881891 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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 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 RDDYQNLVSA 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

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMSodium chlorideNaClSodium chloride
1.5 mMtris(2-carboxyethyl)phosphine
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 30.0 kPa / Details: 25 mA current was applied to grid.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 4.5 seconds before plunging..
DetailsThis sample was monodisperse

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Number grids imaged: 1 / Number real images: 1500 / Average electron dose: 43.0 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 362627
CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER / Details: ab initio model generated in cryoSPARC
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: COMMON LINE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.5.1)
Details: Following final reconstruction, sharpening was applied using DeepEMhancer. Reconstruction before sharpening is supplied as an additional map.
Number images used: 94974
FSC plot (resolution estimation)

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