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- EMDB-35306: Cryo-EM structure of the yeast SPT-ORM2 (ORM2-S3A-N71A) complex -

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Basic information

Entry
Database: EMDB / ID: EMD-35306
TitleCryo-EM structure of the yeast SPT-ORM2 (ORM2-S3A-N71A) complex
Map data
Sample
  • Complex: SPT-ORM2 complex
    • Protein or peptide: chimera of Long chain base biosynthesis protein 1 and Serine palmitoyltransferase 1
    • Protein or peptide: Serine palmitoyltransferase 2
    • Protein or peptide: Protein ORM2
    • Protein or peptide: Serine palmitoyltransferase-regulating protein TSC3
  • Ligand: PYRIDOXAL-5'-PHOSPHATE
Keywordsceramide / phosphorylation / TRANSFERASE-INHIBITOR COMPLEX
Function / homology
Function and homology information


positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / negative regulation of sphingolipid biosynthetic process / multidimensional cell growth / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / : / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / ceramide metabolic process ...positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / negative regulation of sphingolipid biosynthetic 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 / enzyme activator activity / Neutrophil degranulation / pyridoxal phosphate binding / endoplasmic reticulum membrane / 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
Serine palmitoyltransferase 1 / Serine palmitoyltransferase 2 / Protein ORM2 / Serine palmitoyltransferase-regulating protein TSC3 / Long chain base biosynthesis protein 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Saccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsXie T / Gong X
Funding support1 items
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, 2023-
Header (metadata) releaseFeb 14, 2024-
Map releaseFeb 14, 2024-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_35306.png

Downloads & links

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Map

FileDownload / File: emd_35306.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: 0.022
Minimum - Maximum-0.09353729 - 0.16908143
Average (Standard dev.)0.00026790885 (±0.0055791745)
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: #1

Fileemd_35306_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_35306_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-ORM2 complex

EntireName: SPT-ORM2 complex
Components
  • Complex: SPT-ORM2 complex
    • Protein or peptide: chimera of Long chain base biosynthesis protein 1 and Serine palmitoyltransferase 1
    • Protein or peptide: Serine palmitoyltransferase 2
    • Protein or peptide: Protein ORM2
    • Protein or peptide: Serine palmitoyltransferase-regulating protein TSC3
  • Ligand: PYRIDOXAL-5'-PHOSPHATE

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Supramolecule #1: SPT-ORM2 complex

SupramoleculeName: SPT-ORM2 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: chimera of Long chain base biosynthesis protein 1 and Serine palm...

MacromoleculeName: chimera of Long chain base biosynthesis protein 1 and Serine palmitoyltransferase 1
type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: serine C-palmitoyltransferase
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast) / Strain: S288c
Molecular weightTheoretical: 59.354672 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MASNLVEMFN AALNWVTMIL ESPSARVVLF GVPIRGHFFV EGLLGVVIII LLTRKSYKPP KRPLTEQEID ELCDEWVPEP LVDPSATDE QSWRVAKTPV TMEMPIQNHI TITRNNLQEK YTNVFNLASN NFLQLSATEP VKEVVKTTIK NYGVGACGPA G FYGNQDVH ...String:
MASNLVEMFN AALNWVTMIL ESPSARVVLF GVPIRGHFFV EGLLGVVIII LLTRKSYKPP KRPLTEQEID ELCDEWVPEP LVDPSATDE QSWRVAKTPV TMEMPIQNHI TITRNNLQEK YTNVFNLASN NFLQLSATEP VKEVVKTTIK NYGVGACGPA G FYGNQDVH YTLEYDLAQF FGTQGSVLYG QDFCAAPSVL PAFTKRGDVI VADDQVSLPV QNALQLSRST VYYFNHNDMN SL ECLLNEL TEQEKLEKLP AIPRKFIVTE GIFHNSGDLA PLPELTKLKN KYKFRLFVDE TFSIGVLGAT GRGLSEHFNM DRA TAIDIT VGSMATALGS TGGFVLGDSV MCLHQRIGSN AYCFSACLPA YTVTSVSKVL KLMDSNNDAV QTLQKLSKSL HDSF ASDDS LRSYVIVTSS PVSAVLHLQL TPAYRSRKFG YTCEQLFETM SALQKKSQTN KFIEPYEEEE KFLQSIVDHA LINYN VLIT RNTIVLKQET LPIVPSLKIC CNAAMSPEEL KNACESVKQS ILACCQESNK

UniProtKB: Long chain base biosynthesis protein 1, Serine palmitoyltransferase 1

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Macromolecule #2: Serine palmitoyltransferase 2

MacromoleculeName: Serine palmitoyltransferase 2 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: serine C-palmitoyltransferase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 63.189707 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MSTPANYTRV PLCEPEELPD DIQKENEYGT LDSPGHLYQV KSRHGKPLPE PVVDTPPYYI SLLTYLNYLI LIILGHVHDF LGMTFQKNK HLDLLEHDGL APWFSNFESF YVRRIKMRID DCFSRPTTGV PGRFIRCIDR ISHNINEYFT YSGAVYPCMN L SSYNYLGF ...String:
MSTPANYTRV PLCEPEELPD DIQKENEYGT LDSPGHLYQV KSRHGKPLPE PVVDTPPYYI SLLTYLNYLI LIILGHVHDF LGMTFQKNK HLDLLEHDGL APWFSNFESF YVRRIKMRID DCFSRPTTGV PGRFIRCIDR ISHNINEYFT YSGAVYPCMN L SSYNYLGF AQSKGQCTDA ALESVDKYSI QSGGPRAQIG TTDLHIKAEK LVARFIGKED ALVFSMGYGT NANLFNAFLD KK CLVISDE LNHTSIRTGV RLSGAAVRTF KHGDMVGLEK LIREQIVLGQ PKTNRPWKKI LICAEGLFSM EGTLCNLPKL VEL KKKYKC YLFIDEAHSI GAMGPTGRGV CEIFGVDPKD VDILMGTFTK SFGAAGGYIA ADQWIIDRLR LDLTTVSYSE SMPA PVLAQ TISSLQTISG EICPGQGTER LQRIAFNSRY LRLALQRLGF IVYGVADSPV IPLLLYCPSK MPAFSRMMLQ RRIAV VVVA YPATPLIESR VRFCMSASLT KEDIDYLLRH VSEVGDKLNL KSNSGKSSYD GKRQRWDIEE VIRRTPEDCK DDKYFV N

UniProtKB: Serine palmitoyltransferase 2

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Macromolecule #3: Protein ORM2

MacromoleculeName: Protein ORM2 / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast) / Strain: S288c
Molecular weightTheoretical: 24.787576 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MIDRTKNESP AFEESPLTPN VSNLKPFPSQ SNKISTPVTD HRRRRAAAVI SHVEQETFED ENDQQMLPNM AATWVDQRGA WLIHIVVIV LLRLFYSLFG STPKWTWTLT NMTYIIGFYI MFHLVKGTPF DFNGGAYDNL TMWEQINDET LYTPTRKFLL I VPIVLFLI ...String:
MIDRTKNESP AFEESPLTPN VSNLKPFPSQ SNKISTPVTD HRRRRAAAVI SHVEQETFED ENDQQMLPNM AATWVDQRGA WLIHIVVIV LLRLFYSLFG STPKWTWTLT NMTYIIGFYI MFHLVKGTPF DFNGGAYDNL TMWEQINDET LYTPTRKFLL I VPIVLFLI SNQYYRNDMT LFLSNLAVTV LIGVVPKLGI THRLRISIPG ITGRAQIS

UniProtKB: Protein ORM2

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Macromolecule #4: Serine palmitoyltransferase-regulating protein TSC3

MacromoleculeName: Serine palmitoyltransferase-regulating protein TSC3 / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 9.590233 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MTQHKSSMVY IPTTKEAKRR NGKSEGILNT IEEVVEKLYW TYYIHLPFYL MASFDSFFLH VFFLTIFSLS FFGILKYCFL

UniProtKB: Serine palmitoyltransferase-regulating protein TSC3

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Macromolecule #5: PYRIDOXAL-5'-PHOSPHATE

MacromoleculeName: PYRIDOXAL-5'-PHOSPHATE / type: ligand / ID: 5 / Number of copies: 2 / Formula: PLP
Molecular weightTheoretical: 247.142 Da
Chemical component information

ChemComp-PLP:
PYRIDOXAL-5'-PHOSPHATE

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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: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 200035
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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