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- PDB-8qof: Cryo-EM structure of the yeast SPT-Orm2-Dimer complex -

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Basic information

Entry
Database: PDB / ID: 8qof
TitleCryo-EM structure of the yeast SPT-Orm2-Dimer complex
Components
  • (Serine palmitoyltransferase ...) x 2
  • ORM2 isoform 1
  • Serine palmitoyltransferase-regulating protein TSC3
KeywordsTRANSFERASE / Serine-Palmitoyl-Transferase
Function / homology
Function and homology information


positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / sphingosine biosynthetic process / sphingolipid biosynthetic process / ceramide biosynthetic process / enzyme activator activity ...positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / sphingosine biosynthetic process / sphingolipid biosynthetic process / ceramide biosynthetic process / enzyme activator activity / pyridoxal phosphate binding / endoplasmic reticulum membrane / endoplasmic reticulum / membrane
Similarity search - Function
: / 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
PYRIDOXAL-5'-PHOSPHATE / Chem-Q7G / : / : / Serine palmitoyltransferase 1 / Serine palmitoyltransferase 2 / 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. / Moeller, A. / Froehlich, F.
Funding support Germany, 1items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB1557 Germany
CitationJournal: Cell Rep / Year: 2024
Title: The structure of the Orm2-containing serine palmitoyltransferase complex reveals distinct inhibitory potentials of yeast Orm proteins.
Authors: Carolin Körner / Jan-Hannes Schäfer / Bianca M Esch / Kristian Parey / Stefan Walter / David Teis / Dovile Januliene / Oliver Schmidt / Arne Moeller / Florian Fröhlich /
Abstract: Sphingolipid levels are crucial determinants of neurodegenerative disorders and therefore require tight regulation. The Orm protein family and ceramides inhibit the rate-limiting step of sphingolipid ...Sphingolipid levels are crucial determinants of neurodegenerative disorders and therefore require tight regulation. The Orm protein family and ceramides inhibit the rate-limiting step of sphingolipid biosynthesis-the condensation of L-serine and palmitoyl-coenzyme A (CoA). The yeast isoforms Orm1 and Orm2 form a complex with the serine palmitoyltransferase (SPT). While Orm1 and Orm2 have highly similar sequences, they are differentially regulated, though the mechanistic details remain elusive. Here, we determine the cryoelectron microscopy structure of the SPT complex containing Orm2. Complementary in vitro activity assays and genetic experiments with targeted lipidomics demonstrate a lower activity of the SPT-Orm2 complex than the SPT-Orm1 complex. Our results suggest a higher inhibitory potential of Orm2, despite the similar structures of the Orm1- and Orm2-containing complexes. The high conservation of SPT from yeast to man implies different regulatory capacities for the three human ORMDL isoforms, which might be key for understanding their role in sphingolipid-mediated neurodegenerative disorders.
History
DepositionSep 28, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 4, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
B: Serine palmitoyltransferase 1
F: Serine palmitoyltransferase 1
D: Serine palmitoyltransferase-regulating protein TSC3
H: Serine palmitoyltransferase-regulating protein TSC3
A: ORM2 isoform 1
E: ORM2 isoform 1
G: Serine palmitoyltransferase 2
C: Serine palmitoyltransferase 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)329,43714
Polymers325,1928
Non-polymers4,2456
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Serine palmitoyltransferase ... , 2 types, 4 molecules BFGC

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


Mass: 64985.520 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: N-FLAG-Tag Insertion (): MAHIPEVLP(DYKDHDGDYKDHDIDYKDDDDK)KSIPIPAFI...
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
#4: Protein Serine palmitoyltransferase 2 / SPT 2 / Long chain base biosynthesis protein 2


Mass: 63189.707 Da / Num. of mol.: 2
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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Protein , 2 types, 4 molecules DHAE

#2: Protein Serine palmitoyltransferase-regulating protein TSC3 / Temperature-sensitive CSG2-mutant suppressor protein 3


Mass: 9590.233 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Tsc3
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TSC3, YBR058C-A / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q3E790
#3: Protein ORM2 isoform 1


Mass: 24830.602 Da / Num. of mol.: 2 / Mutation: S46A, S47A, S48A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: ORM2 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: A0A6L0ZQC3

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Non-polymers , 3 types, 6 molecules

#5: Chemical ChemComp-WAR / ~{N}-[(2~{S},3~{S},4~{R})-1,3,4-tris(oxidanyl)octadecan-2-yl]heptacosanamide


Mass: 710.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C45H91NO4 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H10NO6P / Feature type: SUBJECT OF INVESTIGATION
#7: 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.: 2 / Source method: obtained synthetically / Formula: C56H92O25 / Feature type: SUBJECT OF INVESTIGATION

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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: C2 symmetric complex of Lcb1, Lcb2, Orm2 and Tsc3 / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightValue: 0.32 MDa / Experimental value: NO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 6.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: TFS FALCON 4i (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 148610 / Symmetry type: POINT

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