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- EMDB-23610: Cryo-EM asymmetric structure of Grafix-stabilized PaFS octameric ... -

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Basic information

Entry
Database: EMDB / ID: EMD-23610
TitleCryo-EM asymmetric structure of Grafix-stabilized PaFS octameric prenyltransferase domain
Map dataOctameric core prenyltransferase domain of PaFS, subjected to crosslinking, with imposed C2 symmetry
Sample
  • Complex: Apo octameric core prenyltransferase domain of PaFS
    • Protein or peptide: Fusicoccadiene synthase from Phomopsis amygdali
Function / homology
Function and homology information


fusicocca-2,10(14)-diene synthase / alcohol biosynthetic process / mycotoxin biosynthetic process / geranylgeranyl diphosphate synthase / ketone biosynthetic process / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 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
DepositionMar 10, 2021-
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: 0.0277
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.0277
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23610.png

Downloads & links

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Map

FileDownload / File: emd_23610.map.gz / Format: CCP4 / Size: 10.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOctameric core prenyltransferase domain of PaFS, subjected to crosslinking, with imposed C2 symmetry
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.16 Å/pix.
x 140 pix.
= 302.4 Å		2.16 Å/pix.
x 140 pix.
= 302.4 Å		2.16 Å/pix.
x 140 pix.
= 302.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.16 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0277 / Movie #1: 0.0277
Minimum - Maximum-0.030401198 - 0.10243007
Average (Standard dev.)0.0003044316 (±0.0046452014)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions140140140
Spacing140140140
CellA=B=C: 302.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.162.162.16
M x/y/z140140140
origin x/y/z0.0000.0000.000
length x/y/z302.400302.400302.400
α/β/γ90.00090.00090.000
start NX/NY/NZ1331310
NX/NY/NZ223226424
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS140140140
D min/max/mean-0.0300.1020.000

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Supplemental data

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Mask #1

Fileemd_23610_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unsharpened, unfiltered half-map, particles subjected to partial signal...

Fileemd_23610_half_map_1.map
Annotationunsharpened, unfiltered half-map, particles subjected to partial signal subtraction
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: unsharpened, unfiltered half-map, particles subjected to partial signal...

Fileemd_23610_half_map_2.map
Annotationunsharpened, unfiltered half-map, particles subjected to partial signal subtraction
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Apo octameric core prenyltransferase domain of PaFS

EntireName: Apo octameric core prenyltransferase domain of PaFS
Components
  • Complex: Apo octameric core prenyltransferase domain of PaFS
    • Protein or peptide: Fusicoccadiene synthase from Phomopsis amygdali

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Supramolecule #1: Apo octameric core prenyltransferase domain of PaFS

SupramoleculeName: Apo octameric core prenyltransferase domain of PaFS / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Reconstruction of single domain of full-length complex was calculated by partial signal subtraction. Refinement imposed C2 symmetry.
Source (natural)Organism: Diaporthe amygdali (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
Molecular weightExperimental: 392 KDa

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Macromolecule #1: Fusicoccadiene synthase from Phomopsis amygdali

MacromoleculeName: Fusicoccadiene synthase from Phomopsis amygdali / type: protein_or_peptide / ID: 1 / 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 PCGEYRGTLG PRFSFISVA VPECIPERLE VISYANEFAF LHDDVTDHVG HDTGEVENDE MMTVFLEAAH T GAIDTSNK VDIRRAGKKR IQSQLFLEML ...String:
MGSSHHHHHH SSGLVPRGSH MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCGEYRGTLG PRFSFISVA VPECIPERLE VISYANEFAF LHDDVTDHVG HDTGEVENDE MMTVFLEAAH T GAIDTSNK VDIRRAGKKR IQSQLFLEML AIDPECAKTT MKSWARFVEV GSSRQHETRF VE LAKYIPY RIMDVGEMFW FGLVTFGLGL HIPDHELELC RELMANAWIA VGLQNDIWSW PKE RDAATL HGKDHVVNAI WVLMQEHQTD VDGAMQICRK LIVEYVAKYL EVIEATKNDE SISL DLRKY LDAMLYSISG NVVWSLECPR YNPDVSFNKT QLEWMRQGLP SLESCPVLAR SPEID SDES AVSPTADESD STEDSLGSGS RQDSSLSTGL SLSPVHSNEG KDLQRVDTDH IFFEKA VLE APYDYIASMP SKGVRDQFID ALNDWLRVPD VKVGKIKDAV RVLHNSSLLL DDFQDNS PL RRGKPSTHNI FGSAQTVNTA TYSIIKAIGQ IMEFSAGESV QEVMNSIMIL FQGQAMDL F WTYNGHVPSE EEYYRMIDQK TGQLFSIATS LLLNAADNEI PRTKIQSCLH RLTRLLGRC FQIRDDYQNL VSADYTKQKG FCEDLDEGKW SLALIHMIHK QRSHMALLNV LSTGRKHGGM TLEQKQFVL DIIEEEKSLD YTRSVMMDLH VQLRAEIGRI EILLDSPNPA MRLLLELLRV

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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: 7.7
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE
DetailsPaFS was subjected to gradient fixation using glutaraldehyde

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / 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

Particle selectionNumber selected: 312022
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 48325
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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