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- PDB-8c81: Cryo-EM structure of the yeast SPT-Orm1-Sac1 complex -

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Basic information

Entry
Database: PDB / ID: 8c81
TitleCryo-EM structure of the yeast SPT-Orm1-Sac1 complex
Components
  • (Serine palmitoyltransferase ...) x 2
  • Phosphatidylinositol-3-phosphatase SAC1
  • Protein ORM1
  • Serine palmitoyltransferase-regulating protein TSC3
KeywordsTRANSFERASE / Serine-Palmitoyl-Transferase / SPT / Orm-Protein
Function / homology
Function and homology information


phosphatidylinositol-3-phosphatase / Synthesis of PIPs at the ER membrane / phosphatidylinositol-4-phosphate phosphatase activity / negative regulation of sphingolipid biosynthetic process / positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / phosphatidylinositol-3,5-bisphosphate 3-phosphatase activity / host cell viral assembly compartment / phosphatidylinositol-3-phosphate phosphatase activity / Synthesis of PIPs at the Golgi membrane ...phosphatidylinositol-3-phosphatase / Synthesis of PIPs at the ER membrane / phosphatidylinositol-4-phosphate phosphatase activity / negative regulation of sphingolipid biosynthetic process / positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / phosphatidylinositol-3,5-bisphosphate 3-phosphatase activity / host cell viral assembly compartment / phosphatidylinositol-3-phosphate phosphatase activity / Synthesis of PIPs at the Golgi membrane / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / cortical endoplasmic reticulum / ceramide metabolic process / autophagosome-lysosome fusion / sphingosine biosynthetic process / sphingolipid biosynthetic process / phosphatidylinositol dephosphorylation / ceramide biosynthetic process / Golgi medial cisterna / response to unfolded protein / Neutrophil degranulation / enzyme activator activity / pyridoxal phosphate binding / mitochondrial outer membrane / Golgi membrane / endoplasmic reticulum membrane / endoplasmic reticulum / mitochondrion / membrane
Similarity search - Function
SAC domain / SacI homology domain / Sac phosphatase domain profile. / ORMDL family / ORMDL family / : / Aminotransferase, class-II, pyridoxal-phosphate binding site / Aminotransferases class-II pyridoxal-phosphate attachment site. / Aminotransferase, class I/classII / Aminotransferase class I and II ...SAC domain / SacI homology domain / Sac phosphatase domain profile. / ORMDL family / ORMDL family / : / Aminotransferase, class-II, pyridoxal-phosphate binding site / Aminotransferases class-II pyridoxal-phosphate attachment site. / Aminotransferase, class I/classII / Aminotransferase class I and II / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
ERGOSTEROL / PYRIDOXAL-5'-PHOSPHATE / Chem-Q7G / Chem-Z8A / Serine palmitoyltransferase 1 / Phosphatidylinositol-3-phosphatase SAC1 / Serine palmitoyltransferase 2 / Protein ORM1 / Serine palmitoyltransferase-regulating protein TSC3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsSchaefer, J. / Koerner, C. / Parey, K. / Januliene, D. / Moeller, A. / Froehlich, F.
Funding support Germany, 2items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB944 Germany
German Research Foundation (DFG)SFB15 Germany
CitationJournal: Nat Commun / Year: 2023
Title: Structure of the ceramide-bound SPOTS complex.
Authors: Jan-Hannes Schäfer / Carolin Körner / Bianca M Esch / Sergej Limar / Kristian Parey / Stefan Walter / Dovile Januliene / Arne Moeller / Florian Fröhlich /
Abstract: Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. ...Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide-dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.
History
DepositionJan 18, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 11, 2023Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Protein ORM1
B: Serine palmitoyltransferase 1
C: Serine palmitoyltransferase 2
D: Serine palmitoyltransferase-regulating protein TSC3
E: Phosphatidylinositol-3-phosphatase SAC1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)237,50011
Polymers234,2015
Non-polymers3,2996
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 3 types, 3 molecules ADE

#1: Protein Protein ORM1


Mass: 25221.674 Da / Num. of mol.: 1 / Mutation: S51A, S52A, S53A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ORM1, YGR038W / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P53224
#4: Protein Serine palmitoyltransferase-regulating protein TSC3 / Temperature-sensitive CSG2-mutant suppressor protein 3


Mass: 9590.233 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TSC3, YBR058C-A / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q3E790
#5: Protein Phosphatidylinositol-3-phosphatase SAC1 / Phosphatidylinositol-4-phosphate phosphatase / Recessive suppressor of secretory defect


Mass: 71214.352 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: SAC1, RSD1, YKL212W / Production host: Saccharomyces cerevisiae (brewer's yeast)
References: UniProt: P32368, phosphatidylinositol-3-phosphatase

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Serine palmitoyltransferase ... , 2 types, 2 molecules BC

#2: Protein Serine palmitoyltransferase 1 / SPT 1 / SPT1 / Long chain base biosynthesis protein 1


Mass: 64985.520 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: LCB1, END8, TSC2, YMR296C / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P25045, serine C-palmitoyltransferase
#3: Protein Serine palmitoyltransferase 2 / SPT 2 / Long chain base biosynthesis protein 2


Mass: 63189.707 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: LCB2, SCS1, TSC1, YDR062W, D4246, YD9609.16 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: P40970, serine C-palmitoyltransferase

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Non-polymers , 4 types, 6 molecules

#6: Chemical ChemComp-ERG / ERGOSTEROL


Mass: 396.648 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C28H44O
#7: Chemical ChemComp-Z8A / N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]hexacosanamide


Mass: 696.182 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C44H89NO4 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical ChemComp-Q7G / 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl}-4-{[(3beta,9beta,14beta,17beta,25R)-spirost-5-en-3-yl]oxy}butyl 4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranoside


Mass: 1165.315 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C56H92O25
#9: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C8H10NO6P

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Heteropentameric complex of Orm1 with Lcb1, Lcb2, Tsc3 and Sac1
Type: COMPLEX / Entity ID: #1-#5 / Source: RECOMBINANT
Molecular weightValue: 0.23 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 6.8
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 13604
EM imaging opticsEnergyfilter name: TFS Selectris / Energyfilter slit width: 10 eV

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Processing

SoftwareName: UCSF ChimeraX / Version: 1.6/v9 / Classification: model building / URL: https://www.rbvi.ucsf.edu/chimerax/ / Os: macOS / Type: package
EM software
IDName
1EPU
2cryoSPARC
3Coot
4PHENIX
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 53236 / Symmetry type: POINT
Atomic model buildingB value: 63 / Protocol: AB INITIO MODEL / Space: REAL
RefinementHighest resolution: 3.3 Å

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