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- EMDB-22487: Cryo-EM structure of Grafix-stabilized PaFS prenyltransferase oct... -

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Basic information

Entry
Database: EMDB / ID: EMD-22487
TitleCryo-EM structure of Grafix-stabilized PaFS prenyltransferase octamer with a peripheral cyclase domain - symmetry expanded class B
Map dataFinal processed map - Symmetry Expanded Class B
Sample
  • Complex: Phomopsis amygdali fusicoccadiene synthase (PaFS)
    • Protein or peptide: Fusicoccadiene synthase
Function / homology
Function and homology information


fusicocca-2,10(14)-diene synthase / alcohol biosynthetic process / mycotoxin biosynthetic process / geranylgeranyl diphosphate synthase / ketone biosynthetic process / farnesyltranstransferase activity / isoprenoid biosynthetic process / lyase activity / metal ion binding
Similarity search - Function
Terpene synthase family 2, C-terminal metal binding / Polyprenyl synthases signature 2. / Polyprenyl synthases signature 1. / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 8.6 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 19, 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: 0.0176
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0176
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22487.png

Downloads & links

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Map

FileDownload / File: emd_22487.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal processed map - Symmetry Expanded Class B
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 192 pix.
= 414.72 Å		2.16 Å/pix.
x 192 pix.
= 414.72 Å		2.16 Å/pix.
x 192 pix.
= 414.72 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 2.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0176 / Movie #1: 0.0176
Minimum - Maximum-0.017275937 - 0.06305986
Average (Standard dev.)0.00028959906 (±0.002946356)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 414.72003 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z192192192
origin x/y/z0.0000.0000.000
length x/y/z414.720414.720414.720
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS192192192
D min/max/mean-0.0170.0630.000

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Supplemental data

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Mask #1

Fileemd_22487_msk_1.map
Projections & Slices
AxesZYX

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Mask #2

Fileemd_22487_msk_2.map
Projections & Slices
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Histogram

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Half map: #1

Fileemd_22487_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_22487_half_map_2.map
Projections & Slices
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Sample components

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Entire : Phomopsis amygdali fusicoccadiene synthase (PaFS)

EntireName: Phomopsis amygdali fusicoccadiene synthase (PaFS)
Components
  • Complex: Phomopsis amygdali fusicoccadiene synthase (PaFS)
    • Protein or peptide: Fusicoccadiene synthase

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Supramolecule #1: Phomopsis amygdali fusicoccadiene synthase (PaFS)

SupramoleculeName: Phomopsis amygdali fusicoccadiene synthase (PaFS) / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: PaFS octameric prenyltransferase domain is well-resolved in structure, with density for a variably positioned cyclase domain alongside the octamer. Molecular weight reflects the calculated ...Details: PaFS octameric prenyltransferase domain is well-resolved in structure, with density for a variably positioned cyclase domain alongside the octamer. Molecular weight reflects the calculated molecular weight of a prenyltransferase octamer plus one cyclase domain.
Source (natural)Organism: Diaporthe amygdali (fungus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria) / Recombinant strain: BL21-CodonPlus (DE3)-RIL
Molecular weightTheoretical: 430 KDa

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Macromolecule #1: Fusicoccadiene synthase

MacromoleculeName: Fusicoccadiene synthase / type: protein_or_peptide / ID: 1
Details: The final reconstruction accounts for an octamer of the C-terminal transferase domain, residues 414-719. Density on the periphery of the octamer accounts for the N-terminal cyclase domain
Enantiomer: LEVO / EC number: fusicocca-2,10(14)-diene synthase
Source (natural)Organism: Diaporthe amygdali (fungus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
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

Concentration0.3 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200
VitrificationCryogen name: ETHANE / Instrument: LEICA EM CPC / Details: Blot for 2.5 seconds before plunging.
DetailsSample was fixed with glutaraldehyde by glycerol gradient centrifugation with a 0-0.125% (v/v) glutaraldehyde gradient. Glycerol was removed by dialysis prior to vitrification.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 6544 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND (ver. 4.1)
Startup modelType of model: OTHER / Details: ab initio model calculated in Relion
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 8.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0.7)
Details: Particles were subjected to symmetry expansion about C4 axis prior to partial signal subtraction (mask provided). Masked 3D classification was performed (mask provided) before final ...Details: Particles were subjected to symmetry expansion about C4 axis prior to partial signal subtraction (mask provided). Masked 3D classification was performed (mask provided) before final reconstruction using entire particles from one selected class (class B).
Number images used: 34583
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: COMMON LINE
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL

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