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- EMDB-6288: Electron cryo-microscopy of fatty acid synthase (FAS) from Rhodos... -

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Basic information

Entry
Database: EMDB / ID: EMD-6288
TitleElectron cryo-microscopy of fatty acid synthase (FAS) from Rhodospiridium toruloides
Map dataFatty acid synthase from Rhodosporidium toruloides
Sample
  • Sample: Fatty acid synthase from Rhodospiridium toruloides
  • Protein or peptide: Fatty acid synthase subunit alpha, fungi type
  • Protein or peptide: Fatty acid synthase subunit beta, fungi type
KeywordsMega-enzyme / multifunctional proteins / protein assembly / acyl carrier protein / biofuel
Function / homology
Function and homology information


: / : / fatty-acyl-CoA synthase system / fatty-acyl-CoA synthase activity / fatty acid synthase complex / (3R)-3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase activity / holo-[acyl-carrier-protein] synthase activity / 3-oxoacyl-[acyl-carrier-protein] reductase / 3-oxoacyl-[acyl-carrier-protein] reductase (NADPH) activity / enoyl-[acyl-carrier-protein] reductase (NADH) activity ...: / : / fatty-acyl-CoA synthase system / fatty-acyl-CoA synthase activity / fatty acid synthase complex / (3R)-3-hydroxypalmitoyl-[acyl-carrier-protein] dehydratase activity / holo-[acyl-carrier-protein] synthase activity / 3-oxoacyl-[acyl-carrier-protein] reductase / 3-oxoacyl-[acyl-carrier-protein] reductase (NADPH) activity / enoyl-[acyl-carrier-protein] reductase (NADH) activity / 3-oxoacyl-[acyl-carrier-protein] synthase activity / fatty acid biosynthetic process / hydrolase activity / magnesium ion binding
Similarity search - Function
: / Fatty acid synthase, meander beta sheet domain / Fatty acid synthase subunit beta, N-terminal domain / N-terminal domain in fatty acid synthase subunit beta / Fatty acid synthase meander beta sheet domain / Fatty acid synthase subunit alpha, acyl carrier domain / Fatty acid synthase subunit alpha Acyl carrier domain / Fatty acid synthase beta subunit AflB /Fas1-like, central domain / Fatty acid synthase beta subunit AflB /Fas1-like, central domain / Fatty acid synthase subunit beta/Fas1-like, helical ...: / Fatty acid synthase, meander beta sheet domain / Fatty acid synthase subunit beta, N-terminal domain / N-terminal domain in fatty acid synthase subunit beta / Fatty acid synthase meander beta sheet domain / Fatty acid synthase subunit alpha, acyl carrier domain / Fatty acid synthase subunit alpha Acyl carrier domain / Fatty acid synthase beta subunit AflB /Fas1-like, central domain / Fatty acid synthase beta subunit AflB /Fas1-like, central domain / Fatty acid synthase subunit beta/Fas1-like, helical / Fatty acid synthase type I, helical / Fatty acid synthase type I helical domain / Fatty acid synthase / Fatty acid synthase / N-terminal of MaoC-like dehydratase / N-terminal half of MaoC dehydratase / Starter unit:ACP transacylase / Starter unit:ACP transacylase in aflatoxin biosynthesis / Holo-[acyl carrier protein] synthase / Holo-[acyl carrier protein] synthase / Phosphopantetheine-protein transferase domain / Phosphopantetheine-protein transferase domain / MaoC-like dehydratase domain / MaoC-like dehydratase domain / MaoC like domain / NAD(P)-binding domain / 4'-phosphopantetheinyl transferase domain / 4'-phosphopantetheinyl transferase domain / 4'-phosphopantetheinyl transferase domain superfamily / 4'-phosphopantetheinyl transferase superfamily / HotDog domain superfamily / HotDog domain superfamily / Acyl transferase domain superfamily / Acyl transferase / Acyl transferase domain superfamily / Acyl transferase / Acyl transferase domain / Acyl transferase domain in polyketide synthase (PKS) enzymes. / Acyl transferase/acyl hydrolase/lysophospholipase / Acyl transferase/acyl hydrolase/lysophospholipase / Beta-ketoacyl synthase, active site / Beta-ketoacyl synthase, active site / Ketosynthase family 3 (KS3) active site signature. / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Beta-ketoacyl synthase, N-terminal / Beta-ketoacyl synthase, C-terminal / Beta-ketoacyl synthase, N-terminal domain / Beta-ketoacyl synthase, C-terminal domain / Thiolase-like / Thiolase-like / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Aldolase-type TIM barrel / Aldolase-type TIM barrel / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Fatty acid synthase subunit beta, fungi type / 3-oxoacyl-[acyl-carrier-protein] reductase
Similarity search - Component
Biological speciesRhodosporidium toruloides (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 7.8 Å
AuthorsFischer M / Rhinow D / Zhu Z / Mills DJ / Zhao ZK / Vonck J / Grininger M
CitationJournal: Protein Sci / Year: 2015
Title: Cryo-EM structure of fatty acid synthase (FAS) from Rhodosporidium toruloides provides insights into the evolutionary development of fungal FAS.
Authors: Manuel Fischer / Daniel Rhinow / Zhiwei Zhu / Deryck J Mills / Zongbao K Zhao / Janet Vonck / Martin Grininger /
Abstract: Fungal fatty acid synthases Type I (FAS I) are up to 2.7 MDa large molecular machines composed of large multifunctional polypeptides. Half of the amino acids in fungal FAS I are involved in ...Fungal fatty acid synthases Type I (FAS I) are up to 2.7 MDa large molecular machines composed of large multifunctional polypeptides. Half of the amino acids in fungal FAS I are involved in structural elements that are responsible for scaffolding the elaborate barrel-shaped architecture and turning fungal FAS I into highly efficient de novo producers of fatty acids. Rhodosporidium toruloides is an oleaginous fungal species and renowned for its robust conversion of carbohydrates into lipids to over 70% of its dry cell weight. Here, we use cryo-EM to determine a 7.8-Å reconstruction of its FAS I that reveals unexpected features; its novel form of splitting the multifunctional polypeptide chain into the two subunits α and β, and its duplicated ACP domains. We show that the specific distribution into α and β occurs by splitting at one of many possible sites that can be accepted by fungal FAS I. While, therefore, the specific distribution in α and β chains in R. toruloides FAS I is not correlated to increased protein activities, we also show that the duplication of ACP is an evolutionary late event and argue that duplication is beneficial for the lipid overproduction phenotype.
History
DepositionFeb 23, 2015-
Header (metadata) releaseMay 27, 2015-
Map releaseMar 23, 2016-
UpdateMar 23, 2016-
Current statusMar 23, 2016Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.07
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.07
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

400_6288.gif

Downloads & links

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Map

FileDownload / File: emd_6288.map.gz / Format: CCP4 / Size: 89 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFatty acid synthase from Rhodosporidium toruloides
Voxel sizeX=Y=Z: 1.32 Å
Density
Contour LevelBy AUTHOR: 0.07 / Movie #1: 0.07
Minimum - Maximum-0.04518283 - 0.17959821
Average (Standard dev.)0.00058107 (±0.01788451)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 380.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.321.321.32
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z380.160380.160380.160
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.0450.1800.001

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Supplemental data

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Sample components

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Entire : Fatty acid synthase from Rhodospiridium toruloides

EntireName: Fatty acid synthase from Rhodospiridium toruloides
Components
  • Sample: Fatty acid synthase from Rhodospiridium toruloides
  • Protein or peptide: Fatty acid synthase subunit alpha, fungi type
  • Protein or peptide: Fatty acid synthase subunit beta, fungi type

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Supramolecule #1000: Fatty acid synthase from Rhodospiridium toruloides

SupramoleculeName: Fatty acid synthase from Rhodospiridium toruloides / type: sample / ID: 1000 / Oligomeric state: a6b6 heterododecameric complex / Number unique components: 2
Molecular weightExperimental: 2.7 MDa / Theoretical: 2.7 MDa

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Macromolecule #1: Fatty acid synthase subunit alpha, fungi type

MacromoleculeName: Fatty acid synthase subunit alpha, fungi type / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Oligomeric state: a6b6 / Recombinant expression: Yes
Source (natural)Organism: Rhodosporidium toruloides (fungus)
Molecular weightTheoretical: 318 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: 3-oxoacyl-[acyl-carrier-protein] reductase
InterPro: 4'-phosphopantetheinyl transferase domain, Acyl transferase domain superfamily, Holo-[acyl carrier protein] synthase, Acyl transferase, Acyl transferase/acyl hydrolase/lysophospholipase, ...InterPro: 4'-phosphopantetheinyl transferase domain, Acyl transferase domain superfamily, Holo-[acyl carrier protein] synthase, Acyl transferase, Acyl transferase/acyl hydrolase/lysophospholipase, Fatty acid synthase, HotDog domain superfamily, Beta-ketoacyl synthase, active site, Beta-ketoacyl synthase, C-terminal, Beta-ketoacyl synthase, N-terminal, MaoC-like dehydratase domain, NAD(P)-binding domain, Phosphopantetheine-protein transferase domain, Thiolase-like, INTERPRO: IPR016038

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Macromolecule #2: Fatty acid synthase subunit beta, fungi type

MacromoleculeName: Fatty acid synthase subunit beta, fungi type / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Oligomeric state: a6b6 / Recombinant expression: Yes
Source (natural)Organism: Rhodosporidium toruloides (fungus)
Molecular weightTheoretical: 137 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceUniProtKB: Fatty acid synthase subunit beta, fungi type
InterPro: Acyl transferase domain superfamily, Acyl transferase/acyl hydrolase/lysophospholipase, Aldolase-type TIM barrel, Fatty acid synthase beta subunit AflB /Fas1-like, central domain, Fatty acid synthase

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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

Concentration1.3 mg/mL
BufferpH: 7.2 / Details: 100 mM sodium phosphate, 200 mM NaCl, 1 mM EDTA
GridDetails: Quantifoil R2/2 holey carbon grids
VitrificationCryogen name: ETHANE / Chamber humidity: 70 % / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 106000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 78000
Sample stageSpecimen holder model: OTHER
Details24-frame movies
DateDec 2, 2013
Image recordingCategory: CCD / Film or detector model: FEI FALCON II (4k x 4k) / Digitization - Sampling interval: 14 µm / Average electron dose: 70 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: Each image
Final reconstructionResolution.type: BY AUTHOR / Resolution: 7.8 Å / Resolution method: OTHER / Software - Name: Relion / Number images used: 3296
DetailsThe reconstruction was done using Relion.

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Atomic model buiding 1

Initial modelPDB ID:

2uvb
PDB Unreleased entry

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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Atomic model buiding 2

Initial modelPDB ID:

2uva
PDB Unreleased entry

SoftwareName: Chimera
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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