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- PDB-8iaj: Cryo-EM structure of the yeast SPT-ORM2 (ORM2-S3A) complex -

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Basic information

Entry
Database: PDB / ID: 8iaj
TitleCryo-EM structure of the yeast SPT-ORM2 (ORM2-S3A) complex
Components
  • Protein ORM2
  • Serine palmitoyltransferase 2
  • Serine palmitoyltransferase-regulating protein TSC3
  • chimera of Long chain base biosynthesis protein 1 and Serine palmitoyltransferase 1
KeywordsTRANSFERASE/INHIBITOR / ceramide / phosphorylation / TRANSFERASE-INHIBITOR COMPLEX
Function / homology
Function and homology information


negative regulation of sphingolipid biosynthetic process / positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / multidimensional cell growth / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / ceramide metabolic process / regulation of programmed cell death ...negative regulation of sphingolipid biosynthetic process / positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / multidimensional cell growth / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / ceramide metabolic process / regulation of programmed cell death / sphingosine biosynthetic process / embryo development ending in seed dormancy / sphingolipid biosynthetic process / ceramide biosynthetic process / response to unfolded protein / Neutrophil degranulation / enzyme activator activity / pyridoxal phosphate binding / endoplasmic reticulum membrane / apoptotic process / endoplasmic reticulum / identical protein binding / membrane
Similarity search - Function
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
PYRIDOXAL-5'-PHOSPHATE / Chem-Z1T / Serine palmitoyltransferase 1 / Serine palmitoyltransferase 2 / Protein ORM2 / Serine palmitoyltransferase-regulating protein TSC3 / Long chain base biosynthesis protein 1
Similarity search - Component
Biological speciesArabidopsis thaliana (thale cress)
Saccharomyces cerevisiae S288C (yeast)
Saccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsXie, T. / Gong, X.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Cell Rep / Year: 2024
Title: Collaborative regulation of yeast SPT-Orm2 complex by phosphorylation and ceramide.
Authors: Tian Xie / Feitong Dong / Gongshe Han / Xinyue Wu / Peng Liu / Zike Zhang / Jianlong Zhong / Somashekarappa Niranjanakumari / Kenneth Gable / Sita D Gupta / Wenchen Liu / Peter J Harrison / ...Authors: Tian Xie / Feitong Dong / Gongshe Han / Xinyue Wu / Peng Liu / Zike Zhang / Jianlong Zhong / Somashekarappa Niranjanakumari / Kenneth Gable / Sita D Gupta / Wenchen Liu / Peter J Harrison / Dominic J Campopiano / Teresa M Dunn / Xin Gong /
Abstract: The homeostatic regulation of serine palmitoyltransferase (SPT) activity in yeast involves N-terminal phosphorylation of Orm proteins, while higher eukaryotes lack these phosphorylation sites. ...The homeostatic regulation of serine palmitoyltransferase (SPT) activity in yeast involves N-terminal phosphorylation of Orm proteins, while higher eukaryotes lack these phosphorylation sites. Although recent studies have indicated a conserved ceramide-mediated feedback inhibition of the SPT-ORM/ORMDL complex in higher eukaryotes, its conservation and relationship with phosphorylation regulation in yeast remain unclear. Here, we determine the structure of the yeast SPT-Orm2 complex in a dephosphomimetic state and identify an evolutionarily conserved ceramide-sensing site. Ceramide stabilizes the dephosphomimetic Orm2 in an inhibitory conformation, facilitated by an intramolecular β-sheet between the N- and C-terminal segments of Orm2. Moreover, we find that a phosphomimetic mutant of Orm2, positioned adjacent to its intramolecular β-sheet, destabilizes the inhibitory conformation of Orm2. Taken together, our findings suggest that both Orm dephosphorylation and ceramide binding are crucial for suppressing SPT activity in yeast. This highlights a distinctive regulatory mechanism in yeast involving the collaborative actions of phosphorylation and ceramide.
History
DepositionFeb 8, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 14, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: chimera of Long chain base biosynthesis protein 1 and Serine palmitoyltransferase 1
B: Serine palmitoyltransferase 2
D: Protein ORM2
C: Serine palmitoyltransferase-regulating protein TSC3
E: chimera of Long chain base biosynthesis protein 1 and Serine palmitoyltransferase 1
F: Serine palmitoyltransferase 2
H: Protein ORM2
G: Serine palmitoyltransferase-regulating protein TSC3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)315,72512
Polymers313,9308
Non-polymers1,7954
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 , 4 types, 8 molecules AEBFDHCG

#1: Protein chimera of Long chain base biosynthesis protein 1 and Serine palmitoyltransferase 1 / AtLCB1 / Protein EMBRYO DEFECTIVE 2779 / Protein FUMONISIN B1 RESISTANT 11 / SPT 1 / SPT1 / Long ...AtLCB1 / Protein EMBRYO DEFECTIVE 2779 / Protein FUMONISIN B1 RESISTANT 11 / SPT 1 / SPT1 / Long chain base biosynthesis protein 1


Mass: 59354.672 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Arabidopsis thaliana (thale cress), (gene. exp.) Saccharomyces cerevisiae S288C
Strain: S288C / Gene: LCB1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human)
References: UniProt: Q94IB8, UniProt: P25045, serine C-palmitoyltransferase
#2: 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 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: P40970, serine C-palmitoyltransferase
#3: Protein Protein ORM2


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 S288C (yeast) / Strain: S288C / Gene: ORM2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q06144
#4: 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
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: TSC3 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q3E790

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Non-polymers , 2 types, 4 molecules

#5: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H10NO6P
#6: Chemical ChemComp-Z1T / N-[(2S,3R,4E)-1,3-dihydroxyoctadec-4-en-2-yl]tetracosanamide


Mass: 650.113 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C42H83NO3 / 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: SPT-ORM2 complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 121303 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00621231
ELECTRON MICROSCOPYf_angle_d0.73428769
ELECTRON MICROSCOPYf_dihedral_angle_d17.3312927
ELECTRON MICROSCOPYf_chiral_restr0.0463256
ELECTRON MICROSCOPYf_plane_restr0.0053651

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