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- EMDB-33866: Cryo-EM structure of SPT-ORMDL3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-33866
TitleCryo-EM structure of SPT-ORMDL3 complex
Map data
Sample
  • Complex: SPT-ORMDL3 complex
    • Protein or peptide: Serine palmitoyltransferase 2
    • Protein or peptide: Serine palmitoyltransferase 1
    • Protein or peptide: ORM1-like protein 3
    • Protein or peptide: Serine palmitoyltransferase small subunit A
  • Protein or peptide: Serine palmitoyltransferase 1
  • Ligand: PYRIDOXAL-5'-PHOSPHATE
  • Ligand: N-[(2S,3R,4E)-1,3-dihydroxyoctadec-4-en-2-yl]tetracosanamide
Keywordsceramide / TRANSFERASE-inhibitor complex / TRANSFERASE-INHIBITOR COMPLEX 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
Serine palmitoyltransferase 1 / Serine palmitoyltransferase 2 / ORM1-like protein 3 / Serine palmitoyltransferase small subunit A
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsXie T / Liu P / Gong X
Funding support1 items
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 18, 2022-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33866.png

Downloads & links

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Map

FileDownload / File: emd_33866.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.027
Minimum - Maximum-0.13370596 - 0.24912259
Average (Standard dev.)0.000010075137 (±0.006322254)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_33866_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_33866_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SPT-ORMDL3 complex

EntireName: SPT-ORMDL3 complex
Components
  • Complex: SPT-ORMDL3 complex
    • Protein or peptide: Serine palmitoyltransferase 2
    • Protein or peptide: Serine palmitoyltransferase 1
    • Protein or peptide: ORM1-like protein 3
    • Protein or peptide: Serine palmitoyltransferase small subunit A
  • Protein or peptide: Serine palmitoyltransferase 1
  • Ligand: PYRIDOXAL-5'-PHOSPHATE
  • Ligand: N-[(2S,3R,4E)-1,3-dihydroxyoctadec-4-en-2-yl]tetracosanamide

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Supramolecule #1: SPT-ORMDL3 complex

SupramoleculeName: SPT-ORMDL3 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Serine palmitoyltransferase 2

MacromoleculeName: Serine palmitoyltransferase 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: serine C-palmitoyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 63.00416 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MRPEPGGCCC RRTVRANGCV ANGEVRNGYV RSSAAAAAAA AAGQIHHVTQ NGGLYKRPFN EAFEETPMLV AVLTYVGYGV LTLFGYLRD FLRYWRIEKC HHATEREEQK DFVSLYQDFE NFYTRNLYMR IRDNWNRPIC SVPGARVDIM ERQSHDYNWS F KYTGNIIK ...String:
MRPEPGGCCC RRTVRANGCV ANGEVRNGYV RSSAAAAAAA AAGQIHHVTQ NGGLYKRPFN EAFEETPMLV AVLTYVGYGV LTLFGYLRD FLRYWRIEKC HHATEREEQK DFVSLYQDFE NFYTRNLYMR IRDNWNRPIC SVPGARVDIM ERQSHDYNWS F KYTGNIIK GVINMGSYNY LGFARNTGSC QEAAAKVLEE YGAGVCSTRQ EIGNLDKHEE LEELVARFLG VEAAMAYGMG FA TNSMNIP ALVGKGCLIL SDELNHASLV LGARLSGATI RIFKHNNMQS LEKLLKDAIV YGQPRTRRPW KKILILVEGI YSM EGSIVR LPEVIALKKK YKAYLYLDEA HSIGALGPTG RGVVEYFGLD PEDVDVMMGT FTKSFGASGG YIGGKKELID YLRT HSHSA VYATSLSPPV VEQIITSMKC IMGQDGTSLG KECVQQLAEN TRYFRRRLKE MGFIIYGNED SPVVPLMLYM PAKIG AFGR EMLKRNIGVV VVGFPATPII ESRARFCLSA AHTKEILDTA LKEIDEVGDL LQLKYSRHRL VPLLDRPFDE TTYEET ED

UniProtKB: Serine palmitoyltransferase 2

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Macromolecule #2: Serine palmitoyltransferase 1

MacromoleculeName: Serine palmitoyltransferase 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO / EC number: serine C-palmitoyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 5.898994 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MATATEQWVL VEMVQALYEA PAYHLILEGI LILWIIRLLF SKTYKLQERS

UniProtKB: Serine palmitoyltransferase 1

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Macromolecule #3: ORM1-like protein 3

MacromoleculeName: ORM1-like protein 3 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 17.512594 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MNVGTAHSEV NPNTRVMNSR GIWLSYVLAI GLLHIVLLSI PFVSVPVVWT LTNLIHNMGM YIFLHTVKGT PFETPDQGKA RLLTHWEQM DYGVQFTASR KFLTITPIVL YFLTSFYTKY DQIHFVLNTV SLMSVLIPKL PQLHGVRIFG INKY

UniProtKB: ORM1-like protein 3

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Macromolecule #4: Serine palmitoyltransferase small subunit A

MacromoleculeName: Serine palmitoyltransferase small subunit A / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 10.742409 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MADYKDDDDK SGPDEVDASG RMAGMALARA WKQMSWFYYQ YLLVTALYML EPWERTVFNS MLVSIVGMAL YTGYVFMPQH IMAILHYFE IVQ

UniProtKB: Serine palmitoyltransferase small subunit A

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Macromolecule #5: Serine palmitoyltransferase 1

MacromoleculeName: Serine palmitoyltransferase 1 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO / EC number: serine C-palmitoyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.925828 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DLTVKEKEEL IEEWQPEPLV PPVPKDHPAL NYNIVSGPPS HKTVVNGKEC INFASFNFLG LLDNPRVKAA ALASLKKYGV GTCGPRGFY GTFDVHLDLE DRLAKFMKTE EAIIYSYGFA TIASAIPAYS KRGDIVFVDR AACFAIQKGL QASRSDIKLF K HNDMADLE ...String:
DLTVKEKEEL IEEWQPEPLV PPVPKDHPAL NYNIVSGPPS HKTVVNGKEC INFASFNFLG LLDNPRVKAA ALASLKKYGV GTCGPRGFY GTFDVHLDLE DRLAKFMKTE EAIIYSYGFA TIASAIPAYS KRGDIVFVDR AACFAIQKGL QASRSDIKLF K HNDMADLE RLLKEQEIED QKNPRKARVT RRFIVVEGLY MNTGTICPLP ELVKLKYKYK ARIFLEESLS FGVLGEHGRG VT EHYGINI DDIDLISANM ENALASIGGF CCGRSFVIDH QRLSGQGYCF SASLPPLLAA AAIEALNIME ENPGIFAVLK EKC GQIHKA LQGISGLKVV GESLSPAFHL QLEESTGSRE QDVRLLQEIV DQCMNRSIAL TQARYLEKEE KCLPPPSIRV VVTV EQTEE ELERAASTIK EVAQAVLL

UniProtKB: Serine palmitoyltransferase 1

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Macromolecule #6: PYRIDOXAL-5'-PHOSPHATE

MacromoleculeName: PYRIDOXAL-5'-PHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: PLP
Molecular weightTheoretical: 247.142 Da
Chemical component information

ChemComp-PLP:
PYRIDOXAL-5'-PHOSPHATE

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Macromolecule #7: N-[(2S,3R,4E)-1,3-dihydroxyoctadec-4-en-2-yl]tetracosanamide

MacromoleculeName: N-[(2S,3R,4E)-1,3-dihydroxyoctadec-4-en-2-yl]tetracosanamide
type: ligand / ID: 7 / Number of copies: 1 / Formula: Z1T
Molecular weightTheoretical: 650.113 Da
Chemical component information

ChemComp-Z1T:
N-[(2S,3R,4E)-1,3-dihydroxyoctadec-4-en-2-yl]tetracosanamide

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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

BufferpH: 7.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 358765
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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