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- EMDB-18770: Structure of human ceramide synthase 6 (CerS6) bound to C16:0 -

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Basic information

Entry
Database: EMDB / ID: EMD-18770
TitleStructure of human ceramide synthase 6 (CerS6) bound to C16:0
Map dataFinal cryoSPARC sharpened map after NU refinement used for model refinement
Sample
  • Complex: CerS6-Nb22 complex
    • Protein or peptide: Isoform 2 of Ceramide synthase 6
    • Protein or peptide: Nanobody-22
  • Ligand: PALMITIC ACID
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
KeywordsCERAMIDE / SPHINGOLIPID / COVALENT INTERMEDIATE / MEMBRANE PROTEIN
Function / homology
Function and homology information


positive regulation of oligodendrocyte apoptotic process / sphingoid base N-palmitoyltransferase / sphingosine N-acyltransferase activity / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / ceramide biosynthetic process / oligodendrocyte development / inflammatory response / endoplasmic reticulum membrane / DNA binding / membrane
Similarity search - Function
TRAM/LAG1/CLN8 homology domain / Sphingosine N-acyltransferase Lag1/Lac1-like / TLC domain / TLC domain profile. / TRAM, LAG1 and CLN8 homology domains. / Homeodomain / Homeobox domain / Homeobox-like domain superfamily
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / Vicugna pacos (alpaca)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsPascoa TC / Pike ACW / Chi G / Stefanic S / Quigley A / Chalk R / Mukhopadhyay SMM / Venkaya S / Dix C / Moreira T ...Pascoa TC / Pike ACW / Chi G / Stefanic S / Quigley A / Chalk R / Mukhopadhyay SMM / Venkaya S / Dix C / Moreira T / Tessitore A / Cole V / Chu A / Elkins JM / Pautsch A / Schnapp G / Carpenter EP / Sauer DB
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Wellcome Trust102164/B/15/Z United Kingdom
Wellcome Trust106169/Z/14/Z United Kingdom
CitationJournal: Nat Struct Mol Biol / Year: 2025
Title: Structural basis of the mechanism and inhibition of a human ceramide synthase.
Authors: Tomas C Pascoa / Ashley C W Pike / Christofer S Tautermann / Gamma Chi / Michael Traub / Andrew Quigley / Rod Chalk / Saša Štefanić / Sven Thamm / Alexander Pautsch / Elisabeth P ...Authors: Tomas C Pascoa / Ashley C W Pike / Christofer S Tautermann / Gamma Chi / Michael Traub / Andrew Quigley / Rod Chalk / Saša Štefanić / Sven Thamm / Alexander Pautsch / Elisabeth P Carpenter / Gisela Schnapp / David B Sauer /
Abstract: Ceramides are bioactive sphingolipids crucial for regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by six ceramide synthase (CerS) enzymes, each with specificity for ...Ceramides are bioactive sphingolipids crucial for regulating cellular metabolism. Ceramides and dihydroceramides are synthesized by six ceramide synthase (CerS) enzymes, each with specificity for different acyl-CoA substrates. Ceramide with a 16-carbon acyl chain (C16 ceramide) has been implicated in obesity, insulin resistance and liver disease and the C16 ceramide-synthesizing CerS6 is regarded as an attractive drug target for obesity-associated disease. Despite their importance, the molecular mechanism underlying ceramide synthesis by CerS enzymes remains poorly understood. Here we report cryo-electron microscopy structures of human CerS6, capturing covalent intermediate and product-bound states. These structures, along with biochemical characterization, reveal that CerS catalysis proceeds through a ping-pong reaction mechanism involving a covalent acyl-enzyme intermediate. Notably, the product-bound structure was obtained upon reaction with the mycotoxin fumonisin B1, yielding insights into its inhibition of CerS. These results provide a framework for understanding CerS function, selectivity and inhibition and open routes for future drug discovery.
History
DepositionOct 26, 2023-
Header (metadata) releaseNov 13, 2024-
Map releaseNov 13, 2024-
UpdateMar 26, 2025-
Current statusMar 26, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18770.png

Downloads & links

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Map

FileDownload / File: emd_18770.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal cryoSPARC sharpened map after NU refinement used for model refinement
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.87 Å/pix.
x 270 pix.
= 236.16 Å		0.87 Å/pix.
x 270 pix.
= 236.16 Å		0.87 Å/pix.
x 270 pix.
= 236.16 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.87467 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.48
Minimum - Maximum-4.201833 - 5.358232
Average (Standard dev.)0.002203198 (±0.11327332)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions270270270
Spacing270270270
CellA=B=C: 236.16008 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18770_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Final cryoSPARC unsharpened map

Fileemd_18770_additional_1.map
AnnotationFinal cryoSPARC unsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap1

Fileemd_18770_half_map_1.map
Annotationhalfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap2

Fileemd_18770_half_map_2.map
Annotationhalfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CerS6-Nb22 complex

EntireName: CerS6-Nb22 complex
Components
  • Complex: CerS6-Nb22 complex
    • Protein or peptide: Isoform 2 of Ceramide synthase 6
    • Protein or peptide: Nanobody-22
  • Ligand: PALMITIC ACID
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose

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Supramolecule #1: CerS6-Nb22 complex

SupramoleculeName: CerS6-Nb22 complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 114.8 KDa

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Macromolecule #1: Isoform 2 of Ceramide synthase 6

MacromoleculeName: Isoform 2 of Ceramide synthase 6 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: sphingoid base N-palmitoyltransferase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 42.418348 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAGILAWFWN ERFWLPHNVT WADLKNTEEA TFPQAEDLYL AFPLAFCIFM VRLIFERFVA KPCAIALNIQ ANGPQIAPPN AILEKVFTA ITKHPDEKRL EGLSKQLDWD VRSIQRWFRQ RRNQEKPSTL TRFCESMWRF SFYLYVFTYG VRFLKKTPWL W NTRHCWYN ...String:
MAGILAWFWN ERFWLPHNVT WADLKNTEEA TFPQAEDLYL AFPLAFCIFM VRLIFERFVA KPCAIALNIQ ANGPQIAPPN AILEKVFTA ITKHPDEKRL EGLSKQLDWD VRSIQRWFRQ RRNQEKPSTL TRFCESMWRF SFYLYVFTYG VRFLKKTPWL W NTRHCWYN YPYQPLTTDL HYYYILELSF YWSLMFSQFT DIKRKDFGIM FLHHLVSIFL ITFSYVNNMA RVGTLVLCLH DS ADALLEA AKMANYAKFQ KMCDLLFVMF AVVFITTRLG IFPLWVLNTT LFESWEIVGP YPSWWVFNLL LLLVQGLNCF WSY LIVKIA CKAVSRGKAG KWNPLHVSKD DRSDAENLYF Q

UniProtKB: Ceramide synthase 6

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Macromolecule #2: Nanobody-22

MacromoleculeName: Nanobody-22 / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Vicugna pacos (alpaca)
Molecular weightTheoretical: 15.070657 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
QVQLVESGGG LVQAEGSLRL SCAASGRTFR TYGMGWFRQA PGKEREFVAA LNWSGSSTYY ADSVKGRFTI SRDNAKNTAY LQMNSLKPE DTAVYYCAAL RRKAEYGSRS IADFDSWSKG TPVTVSSHHH HHHEPEA

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Macromolecule #3: PALMITIC ACID

MacromoleculeName: PALMITIC ACID / type: ligand / ID: 3 / Number of copies: 2 / Formula: PLM
Molecular weightTheoretical: 256.424 Da
Chemical component information

ChemComp-PLM:
PALMITIC ACID

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Macromolecule #4: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 4 / Number of copies: 2 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

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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

Concentration5 mg/mL
BufferpH: 7.5
Component:
ConcentrationName
20.0 mMHEPES
200.0 mMsodium chloride
0.01 % (w/v)glyco-diosgenin

Details: 20 mM HEPES pH 7.5, 200 mM NaCl, and 0.01 % (w/v) GDN
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsSEC-purified

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 14309 / Average exposure time: 1.34 sec. / Average electron dose: 56.36 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3400118
Startup modelType of model: OTHER / Details: ab-initio cryoSPARC
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: non-uniform refinement / Number images used: 93680
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1) / Software - details: non-uniform refinement
Final 3D classificationNumber classes: 10 / Software - Name: RELION (ver. 3.1.3)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-8qz6:
Structure of human ceramide synthase 6 (CerS6) bound to C16:0

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