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- PDB-7yj2: Cryo-EM structure of SPT-ORMDL3 (ORMDL3-N13A) complex -

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Basic information

Entry
Database: PDB / ID: 7yj2
TitleCryo-EM structure of SPT-ORMDL3 (ORMDL3-N13A) complex
Components
  • (Serine palmitoyltransferase ...) x 4
  • ORM1-like protein 3
KeywordsTRANSFERASE/INHIBITOR / ceramide / TRANSFERASE-inhibitor complex
Function / homology
Function and homology information


negative regulation of ceramide biosynthetic process / sphinganine biosynthetic process / regulation of fat cell apoptotic process / sphingomyelin biosynthetic process / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / : / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / ceramide metabolic process ...negative regulation of ceramide biosynthetic process / sphinganine biosynthetic process / regulation of fat cell apoptotic process / sphingomyelin biosynthetic process / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / : / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / ceramide metabolic process / regulation of smooth muscle contraction / sphingosine biosynthetic process / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / sphingolipid metabolic process / ceramide biosynthetic process / negative regulation of B cell apoptotic process / motor behavior / positive regulation of lipophagy / adipose tissue development / positive regulation of autophagy / specific granule membrane / myelination / secretory granule membrane / protein localization / positive regulation of protein localization to nucleus / pyridoxal phosphate binding / Neutrophil degranulation / endoplasmic reticulum membrane / endoplasmic reticulum / plasma membrane
Similarity search - Function
Small subunit of serine palmitoyltransferase-like / Small subunit of serine palmitoyltransferase-like / 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 / Serine palmitoyltransferase 1 / Serine palmitoyltransferase 2 / ORM1-like protein 3 / Serine palmitoyltransferase small subunit A
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsXie, T. / Liu, P. / Gong, X.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2023
Title: Ceramide sensing by human SPT-ORMDL complex for establishing sphingolipid homeostasis.
Authors: Tian Xie / Peng Liu / Xinyue Wu / Feitong Dong / Zike Zhang / Jian Yue / Usha Mahawar / Faheem Farooq / Hisham Vohra / Qi Fang / Wenchen Liu / Binks W Wattenberg / Xin Gong /
Abstract: The ORM/ORMDL family proteins function as regulatory subunits of the serine palmitoyltransferase (SPT) complex, which is the initiating and rate-limiting enzyme in sphingolipid biosynthesis. This ...The ORM/ORMDL family proteins function as regulatory subunits of the serine palmitoyltransferase (SPT) complex, which is the initiating and rate-limiting enzyme in sphingolipid biosynthesis. This complex is tightly regulated by cellular sphingolipid levels, but the sphingolipid sensing mechanism is unknown. Here we show that purified human SPT-ORMDL complexes are inhibited by the central sphingolipid metabolite ceramide. We have solved the cryo-EM structure of the SPT-ORMDL3 complex in a ceramide-bound state. Structure-guided mutational analyses reveal the essential function of this ceramide binding site for the suppression of SPT activity. Structural studies indicate that ceramide can induce and lock the N-terminus of ORMDL3 into an inhibitory conformation. Furthermore, we demonstrate that childhood amyotrophic lateral sclerosis (ALS) variants in the SPTLC1 subunit cause impaired ceramide sensing in the SPT-ORMDL3 mutants. Our work elucidates the molecular basis of ceramide sensing by the SPT-ORMDL complex for establishing sphingolipid homeostasis and indicates an important role of impaired ceramide sensing in disease development.
History
DepositionJul 19, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 5, 2023Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2024Group: Data collection / Source and taxonomy / Category: chem_comp_atom / chem_comp_bond / entity_src_gen

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: Serine palmitoyltransferase 2
E: Serine palmitoyltransferase 1
D: ORM1-like protein 3
C: Serine palmitoyltransferase small subunit A
A: Serine palmitoyltransferase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)144,2886
Polymers144,0415
Non-polymers2471
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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Serine palmitoyltransferase ... , 4 types, 4 molecules BECA

#1: Protein Serine palmitoyltransferase 2 / Serine-palmitoyl-CoA transferase 2 / SPT 2


Mass: 63004.160 Da / Num. of mol.: 1 / Mutation: N13A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPTLC2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: O15270, serine C-palmitoyltransferase
#2: Protein/peptide Serine palmitoyltransferase 1 / Long chain base biosynthesis protein 1 / LCB 1 / Serine-palmitoyl-CoA transferase 1 / SPT 1 / SPT1


Mass: 5898.994 Da / Num. of mol.: 1 / Fragment: UNP residues 1-50
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPTLC1, LCB1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: O15269, serine C-palmitoyltransferase
#4: Protein Serine palmitoyltransferase small subunit A / Small subunit of serine palmitoyltransferase A / ssSPTa


Mass: 10742.409 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPTSSA, C14orf147, SSSPTA / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q969W0
#5: Protein Serine palmitoyltransferase 1 / Long chain base biosynthesis protein 1 / LCB 1 / Serine-palmitoyl-CoA transferase 1 / SPT 1 / SPT1


Mass: 46925.828 Da / Num. of mol.: 1 / Fragment: UNP residues 51-473
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SPTLC1, LCB1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: O15269, serine C-palmitoyltransferase

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Protein / Non-polymers , 2 types, 2 molecules D

#3: Protein ORM1-like protein 3


Mass: 17469.568 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ORMDL3 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8N138
#6: Chemical ChemComp-PLP / PYRIDOXAL-5'-PHOSPHATE / VITAMIN B6 Phosphate


Mass: 247.142 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C8H10NO6P

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Details

Has ligand of interestN

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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-ORMDL3 complex / Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
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: 1.18.1_3865: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 249589 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.019372
ELECTRON MICROSCOPYf_angle_d0.91112687
ELECTRON MICROSCOPYf_dihedral_angle_d26.8111270
ELECTRON MICROSCOPYf_chiral_restr0.061419
ELECTRON MICROSCOPYf_plane_restr0.0061609

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