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- EMDB-16469: Cryo-EM structure of the yeast SPT-Orm1-Dimer complex -

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Basic information

Entry
Database: EMDB / ID: EMD-16469
TitleCryo-EM structure of the yeast SPT-Orm1-Dimer complex
Map dataMap-Dimer
Sample
  • Complex: Complex of Orm1 with Lcb1, Lcb2 and Tsc3
    • Protein or peptide: Protein ORM1
    • Protein or peptide: Serine palmitoyltransferase 1
    • Protein or peptide: Serine palmitoyltransferase 2
    • Protein or peptide: Serine palmitoyltransferase-regulating protein TSC3
  • Ligand: N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]hexacosanamide
  • Ligand: PYRIDOXAL-5'-PHOSPHATE
  • Ligand: 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
KeywordsSerine-Palmitoyl-Transferase / SPT / Orm-Protein / TRANSFERASE
Function / homology
Function and homology information


negative regulation of sphingolipid biosynthetic process / positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / ceramide metabolic process / sphingosine biosynthetic process / sphingolipid biosynthetic process ...negative regulation of sphingolipid biosynthetic process / positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / ceramide metabolic process / sphingosine biosynthetic process / sphingolipid biosynthetic process / ceramide biosynthetic process / response to unfolded protein / Neutrophil degranulation / enzyme activator activity / pyridoxal phosphate binding / endoplasmic reticulum membrane / endoplasmic reticulum / 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 ORM1 / Serine palmitoyltransferase-regulating protein TSC3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsSchaefer J / Koerner C / Parey K / Januliene D / Moeller A / Froehlich F
Funding support Germany, 2 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB944 Germany
German Research Foundation (DFG)SFB1557 Germany
CitationJournal: Nat Commun / Year: 2023
Title: Structure of the ceramide-bound SPOTS complex.
Authors: Jan-Hannes Schäfer / Carolin Körner / Bianca M Esch / Sergej Limar / Kristian Parey / Stefan Walter / Dovile Januliene / Arne Moeller / Florian Fröhlich /
Abstract: Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. ...Sphingolipids are structural membrane components that also function in cellular stress responses. The serine palmitoyltransferase (SPT) catalyzes the rate-limiting step in sphingolipid biogenesis. Its activity is tightly regulated through multiple binding partners, including Tsc3, Orm proteins, ceramides, and the phosphatidylinositol-4-phosphate (PI4P) phosphatase Sac1. The structural organization and regulatory mechanisms of this complex are not yet understood. Here, we report the high-resolution cryo-EM structures of the yeast SPT in complex with Tsc3 and Orm1 (SPOT) as dimers and monomers and a monomeric complex further carrying Sac1 (SPOTS). In all complexes, the tight interaction of the downstream metabolite ceramide and Orm1 reveals the ceramide-dependent inhibition. Additionally, observation of ceramide and ergosterol binding suggests a co-regulation of sphingolipid biogenesis and sterol metabolism within the SPOTS complex.
History
DepositionJan 18, 2023-
Header (metadata) releaseOct 11, 2023-
Map releaseOct 11, 2023-
UpdateOct 11, 2023-
Current statusOct 11, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16469.png

Downloads & links

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Map

FileDownload / File: emd_16469.map.gz / Format: CCP4 / Size: 307.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap-Dimer
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 432 pix.
= 399.168 Å		0.92 Å/pix.
x 432 pix.
= 399.168 Å		0.92 Å/pix.
x 432 pix.
= 399.168 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.924 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.13
Minimum - Maximum-0.92881656 - 1.3970312
Average (Standard dev.)0.0001838157 (±0.025894796)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions432432432
Spacing432432432
CellA=B=C: 399.168 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half-A-Map-Dimer

Fileemd_16469_half_map_1.map
AnnotationHalf-A-Map-Dimer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-B-Map-Dimer

Fileemd_16469_half_map_2.map
AnnotationHalf-B-Map-Dimer
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of Orm1 with Lcb1, Lcb2 and Tsc3

EntireName: Complex of Orm1 with Lcb1, Lcb2 and Tsc3
Components
  • Complex: Complex of Orm1 with Lcb1, Lcb2 and Tsc3
    • Protein or peptide: Protein ORM1
    • Protein or peptide: Serine palmitoyltransferase 1
    • Protein or peptide: Serine palmitoyltransferase 2
    • Protein or peptide: Serine palmitoyltransferase-regulating protein TSC3
  • Ligand: N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]hexacosanamide
  • Ligand: PYRIDOXAL-5'-PHOSPHATE
  • Ligand: 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

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Supramolecule #1: Complex of Orm1 with Lcb1, Lcb2 and Tsc3

SupramoleculeName: Complex of Orm1 with Lcb1, Lcb2 and Tsc3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Details: C2-symmetric complex of Orm1 with Lcb1, Lcb2 and Tsc3 in each protomer
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 325 KDa

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Macromolecule #1: Protein ORM1

MacromoleculeName: Protein ORM1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 25.221674 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MTELDYQGTA EAASTSYSRN QTDLKPFPSA GSASSSIKTT EPVKDHRRRR AAAIISHVEP ETFEDENDQQ LLPNMNATWV DQRGAWIIH VVIIILLKLF YNLFPGVTTE WSWTLTNMTY VIGSYVMFHL IKGTPFDFNG GAYDNLTMWE QIDDETLYTP S RKFLISVP ...String:
MTELDYQGTA EAASTSYSRN QTDLKPFPSA GSASSSIKTT EPVKDHRRRR AAAIISHVEP ETFEDENDQQ LLPNMNATWV DQRGAWIIH VVIIILLKLF YNLFPGVTTE WSWTLTNMTY VIGSYVMFHL IKGTPFDFNG GAYDNLTMWE QIDDETLYTP S RKFLISVP IALFLVSTHY AHYDLKLFSW NCFLTTFGAV VPKLPVTHRL RISIPGITGR AQIS

UniProtKB: Protein ORM1

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Macromolecule #2: Serine palmitoyltransferase 1

MacromoleculeName: Serine palmitoyltransferase 1 / 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: 62.266793 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAHIPEVLPK SIPIPAFIVT TSSYLWYYFN LVLTQIPGGQ FIVSYIKKSH HDDPYRTTVE IGLILYGIIY YLSKPQQKKS LQAQKPNLS PQEIDALIED WEPEPLVDPS ATDEQSWRVA KTPVTMEMPI QNHITITRNN LQEKYTNVFN LASNNFLQLS A TEPVKEVV ...String:
MAHIPEVLPK SIPIPAFIVT TSSYLWYYFN LVLTQIPGGQ FIVSYIKKSH HDDPYRTTVE IGLILYGIIY YLSKPQQKKS LQAQKPNLS PQEIDALIED WEPEPLVDPS ATDEQSWRVA KTPVTMEMPI QNHITITRNN LQEKYTNVFN LASNNFLQLS A TEPVKEVV KTTIKNYGVG ACGPAGFYGN QDVHYTLEYD LAQFFGTQGS VLYGQDFCAA PSVLPAFTKR GDVIVADDQV SL PVQNALQ LSRSTVYYFN HNDMNSLECL LNELTEQEKL EKLPAIPRKF IVTEGIFHNS GDLAPLPELT KLKNKYKFRL FVD ETFSIG VLGATGRGLS EHFNMDRATA IDITVGSMAT ALGSTGGFVL GDSVMCLHQR IGSNAYCFSA CLPAYTVTSV SKVL KLMDS NNDAVQTLQK LSKSLHDSFA SDDSLRSYVI VTSSPVSAVL HLQLTPAYRS RKFGYTCEQL FETMSALQKK SQTNK FIEP YEEEEKFLQS IVDHALINYN VLITRNTIVL KQETLPIVPS LKICCNAAMS PEELKNACES VKQSILACCQ ESNK

UniProtKB: Serine palmitoyltransferase 1

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Macromolecule #3: Serine palmitoyltransferase 2

MacromoleculeName: Serine palmitoyltransferase 2 / type: protein_or_peptide / ID: 3 / 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: Saccharomyces cerevisiae (brewer's yeast)
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 #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: Saccharomyces cerevisiae (brewer's yeast)
SequenceString:
MTQHKSSMVY IPTTKEAKRR NGKSEGILNT IEEVVEKLYW TYYIHLPFYL MASFDSFFLH VFFLTIFSLS FFGILKYCFL

UniProtKB: Serine palmitoyltransferase-regulating protein TSC3

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Macromolecule #5: N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]hexacosanamide

MacromoleculeName: N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]hexacosanamide
type: ligand / ID: 5 / Number of copies: 2 / Formula: Z8A
Molecular weightTheoretical: 696.182 Da
Chemical component information

ChemComp-Z8A:
N-[(2S,3S,4R)-1,3,4-trihydroxyoctadecan-2-yl]hexacosanamide

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Macromolecule #6: PYRIDOXAL-5'-PHOSPHATE

MacromoleculeName: PYRIDOXAL-5'-PHOSPHATE / type: ligand / ID: 6 / Number of copies: 2 / Formula: PLP
Molecular weightTheoretical: 247.142 Da
Chemical component information

ChemComp-PLP:
PYRIDOXAL-5'-PHOSPHATE

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Macromolecule #7: 2-{[(4-O-alpha-D-glucopyranosyl-alpha-D-glucopyranosyl)oxy]methyl...

MacromoleculeName: 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
type: ligand / ID: 7 / Number of copies: 2 / Formula: Q7G
Molecular weightTheoretical: 1.165315 KDa
Chemical component information

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

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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

Concentration10.0 mg/mL
BufferpH: 6.8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Specialist opticsEnergy filter - Name: TFS Selectris / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 13604 / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 141900
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 105
Output model

PDB-8c82:
Cryo-EM structure of the yeast SPT-Orm1-Dimer complex

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