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- EMDB-18537: Cryo-EM structure of the yeast SPT-Orm2-Monomer complex -

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Basic information

Entry
Database: EMDB / ID: EMD-18537
TitleCryo-EM structure of the yeast SPT-Orm2-Monomer complex
Map data
Sample
  • Complex: C2 symmetric complex of Lcb1, Lcb2, Orm2 and Tsc3
    • Protein or peptide: ORM2 isoform 1
    • Protein or peptide: Serine palmitoyltransferase 1
    • Protein or peptide: Serine palmitoyltransferase 2
    • Protein or peptide: Serine palmitoyltransferase-regulating protein TSC3
  • Ligand: ~{N}-[(2~{S},3~{S},4~{R})-1,3,4-tris(oxidanyl)octadecan-2-yl]heptacosanamide
  • 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 / TRANSFERASE
Function / homology
Function and homology information


positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / sphingosine biosynthetic process / sphingolipid biosynthetic process / ceramide biosynthetic process / enzyme activator activity ...positive regulation of sphingolipid biosynthetic process / 3-keto-sphinganine metabolic process / serine palmitoyltransferase complex / intracellular sphingolipid homeostasis / serine C-palmitoyltransferase activity / serine C-palmitoyltransferase / sphingosine biosynthetic process / sphingolipid biosynthetic process / ceramide biosynthetic process / enzyme activator activity / pyridoxal phosphate binding / endoplasmic reticulum membrane / endoplasmic reticulum / membrane
Similarity search - Function
: / 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 / Serine palmitoyltransferase-regulating protein TSC3
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSchaefer J / Koerner C / Moeller A / Froehlich F
Funding support Germany, 1 items
OrganizationGrant numberCountry
German Research Foundation (DFG)SFB1557 Germany
CitationJournal: Cell Rep / Year: 2024
Title: The structure of the Orm2-containing serine palmitoyltransferase complex reveals distinct inhibitory potentials of yeast Orm proteins.
Authors: Carolin Körner / Jan-Hannes Schäfer / Bianca M Esch / Kristian Parey / Stefan Walter / David Teis / Dovile Januliene / Oliver Schmidt / Arne Moeller / Florian Fröhlich /
Abstract: Sphingolipid levels are crucial determinants of neurodegenerative disorders and therefore require tight regulation. The Orm protein family and ceramides inhibit the rate-limiting step of sphingolipid ...Sphingolipid levels are crucial determinants of neurodegenerative disorders and therefore require tight regulation. The Orm protein family and ceramides inhibit the rate-limiting step of sphingolipid biosynthesis-the condensation of L-serine and palmitoyl-coenzyme A (CoA). The yeast isoforms Orm1 and Orm2 form a complex with the serine palmitoyltransferase (SPT). While Orm1 and Orm2 have highly similar sequences, they are differentially regulated, though the mechanistic details remain elusive. Here, we determine the cryoelectron microscopy structure of the SPT complex containing Orm2. Complementary in vitro activity assays and genetic experiments with targeted lipidomics demonstrate a lower activity of the SPT-Orm2 complex than the SPT-Orm1 complex. Our results suggest a higher inhibitory potential of Orm2, despite the similar structures of the Orm1- and Orm2-containing complexes. The high conservation of SPT from yeast to man implies different regulatory capacities for the three human ORMDL isoforms, which might be key for understanding their role in sphingolipid-mediated neurodegenerative disorders.
History
DepositionSep 28, 2023-
Header (metadata) releaseSep 4, 2024-
Map releaseSep 4, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18537.png

Downloads & links

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Map

FileDownload / File: emd_18537.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.92 Å/pix.
x 320 pix.
= 295.68 Å		0.92 Å/pix.
x 320 pix.
= 295.68 Å		0.92 Å/pix.
x 320 pix.
= 295.68 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.924 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-1.5145419 - 1.8501943
Average (Standard dev.)0.002983874 (±0.04357892)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 295.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18537_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_18537_half_map_1.map
Projections & Slices
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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_18537_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : C2 symmetric complex of Lcb1, Lcb2, Orm2 and Tsc3

EntireName: C2 symmetric complex of Lcb1, Lcb2, Orm2 and Tsc3
Components
  • Complex: C2 symmetric complex of Lcb1, Lcb2, Orm2 and Tsc3
    • Protein or peptide: ORM2 isoform 1
    • Protein or peptide: Serine palmitoyltransferase 1
    • Protein or peptide: Serine palmitoyltransferase 2
    • Protein or peptide: Serine palmitoyltransferase-regulating protein TSC3
  • Ligand: ~{N}-[(2~{S},3~{S},4~{R})-1,3,4-tris(oxidanyl)octadecan-2-yl]heptacosanamide
  • 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: C2 symmetric complex of Lcb1, Lcb2, Orm2 and Tsc3

SupramoleculeName: C2 symmetric complex of Lcb1, Lcb2, Orm2 and Tsc3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 160 KDa

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Macromolecule #1: ORM2 isoform 1

MacromoleculeName: ORM2 isoform 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 24.830602 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MIDRTKNESP AFEESPLTPN VSNLKPFPSQ SNKISTPVTD HRRRRAAAVI SHVEQETFED ENDQQMLPNM NATWVDQRGA WLIHIVVIV LLRLFYSLFG STPKWTWTLT NMTYIIGFYI MFHLVKGTPF DFNGGAYDNL TMWEQINDET LYTPTRKFLL I VPIVLFLI ...String:
MIDRTKNESP AFEESPLTPN VSNLKPFPSQ SNKISTPVTD HRRRRAAAVI SHVEQETFED ENDQQMLPNM NATWVDQRGA WLIHIVVIV LLRLFYSLFG STPKWTWTLT NMTYIIGFYI MFHLVKGTPF DFNGGAYDNL TMWEQINDET LYTPTRKFLL I VPIVLFLI SNQYYRNDMT LFLSNLAVTV LIGVVPKLGI THRLRISIPG ITGRAQIS

UniProtKB: UNIPROTKB: A0A6L0ZQC3

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Macromolecule #2: Serine palmitoyltransferase 1

MacromoleculeName: Serine palmitoyltransferase 1 / type: protein_or_peptide / ID: 2
Details: Insertion (N-FLAG-Tag): MAHIPEVLP(DYKDHDGDYKDHDIDYKDDDDK)KSIPIPAFI...
Number of copies: 1 / Enantiomer: LEVO / EC number: serine C-palmitoyltransferase
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 64.98552 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: MAHIPEVLPD YKDHDGDYKD HDIDYKDDDD KKSIPIPAFI VTTSSYLWYY FNLVLTQIPG GQFIVSYIKK SHHDDPYRTT VEIGLILYG IIYYLSKPQQ KKSLQAQKPN LSPQEIDALI EDWEPEPLVD PSATDEQSWR VAKTPVTMEM PIQNHITITR N NLQEKYTN ...String:
MAHIPEVLPD YKDHDGDYKD HDIDYKDDDD KKSIPIPAFI VTTSSYLWYY FNLVLTQIPG GQFIVSYIKK SHHDDPYRTT VEIGLILYG IIYYLSKPQQ KKSLQAQKPN LSPQEIDALI EDWEPEPLVD PSATDEQSWR VAKTPVTMEM PIQNHITITR N NLQEKYTN VFNLASNNFL QLSATEPVKE VVKTTIKNYG VGACGPAGFY GNQDVHYTLE YDLAQFFGTQ GSVLYGQDFC AA PSVLPAF TKRGDVIVAD DQVSLPVQNA LQLSRSTVYY FNHNDMNSLE CLLNELTEQE KLEKLPAIPR KFIVTEGIFH NSG DLAPLP ELTKLKNKYK FRLFVDETFS IGVLGATGRG LSEHFNMDRA TAIDITVGSM ATALGSTGGF VLGDSVMCLH QRIG SNAYC FSACLPAYTV TSVSKVLKLM DSNNDAVQTL QKLSKSLHDS FASDDSLRSY VIVTSSPVSA VLHLQLTPAY RSRKF GYTC EQLFETMSAL QKKSQTNKFI EPYEEEEKFL QSIVDHALIN YNVLITRNTI VLKQETLPIV PSLKICCNAA MSPEEL KNA CESVKQSILA CCQESNK

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: 1 / 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: 1 / 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}-[(2~{S},3~{S},4~{R})-1,3,4-tris(oxidanyl)octadecan-2-yl]hept...

MacromoleculeName: ~{N}-[(2~{S},3~{S},4~{R})-1,3,4-tris(oxidanyl)octadecan-2-yl]heptacosanamide
type: ligand / ID: 5 / Number of copies: 1 / Formula: WAR
Molecular weightTheoretical: 710.208 Da

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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: 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: 1 / 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

BufferpH: 6.5
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: TFS FALCON 4i (4k x 4k) / 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

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71624
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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