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- EMDB-23602: Cryo-EM structure of unliganded octameric prenyltransferase domai... -

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Basic information

Entry
Database: EMDB / ID: EMD-23602
TitleCryo-EM structure of unliganded octameric prenyltransferase domain of Phomopsis amygdali fusicoccadiene synthase, reconstruction in C1
Map dataUnfiltered, sharpened, asymmetric reconstruction of PaFS prenyltransferase domain
Sample
  • Complex: Unliganded Phomopsis amygdali fusicoccadiene synthase octamer
    • 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 / 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 speciesPhomopsis amygdali (fungus) / Diaporthe amygdali (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.18 Å
AuthorsFaylo JL / van Eeuwen T
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 8, 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: 0.141
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.141
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_23602.png

Downloads & links

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Map

FileDownload / File: emd_23602.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnfiltered, sharpened, asymmetric reconstruction of PaFS prenyltransferase domain
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.14 Å/pix.
x 384 pix.
= 438.528 Å		1.14 Å/pix.
x 384 pix.
= 438.528 Å		1.14 Å/pix.
x 384 pix.
= 438.528 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.142 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.141 / Movie #1: 0.141
Minimum - Maximum-0.011439028 - 1.8171797
Average (Standard dev.)0.0005156469 (±0.016260063)
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-0.0111.8170.001

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Supplemental data

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Mask #1

Fileemd_23602_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened, unfiltered, asymmetric reconstruction of PaFS prenyltransferase domain...

Fileemd_23602_additional_1.map
AnnotationUnsharpened, unfiltered, asymmetric reconstruction of PaFS prenyltransferase domain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map of PaFS prenyltransferase domain

Fileemd_23602_half_map_1.map
Annotationhalf-map of PaFS prenyltransferase domain
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half-map of PaFS prenyltransferase domain

Fileemd_23602_half_map_2.map
Annotationhalf-map of PaFS prenyltransferase domain
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 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 / Enantiomer: LEVO / EC number: fusicocca-2,10(14)-diene synthase
Source (natural)Organism: Diaporthe amygdali (fungus)
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MEFKYSEVVE PSTYYTEGLC EGIDVRKSKF TTLEDRGAIR AHEDWNKHIG PCGEYRGTLG PRFSFISVA VPECIPERLE VISYANEFAF LHDDVTDHVG HDTGEVENDE MMTVFLEAAH T GAIDTSNK VDIRRAGKKR IQSQLFLEML AIDPECAKTT MKSWARFVEV ...String:
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: 7.5
Component:
ConcentrationNameFormula
50.0 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
150.0 mMSodium chlorideNaCl
1.5 mMtris(2-carboxyethyl)phosphine
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
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
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
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.18 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.15) / Number images used: 94974
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: COMMON LINE
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7jth

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