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- PDB-8qz7: Structure of human ceramide synthase 6 (CerS6) in complex with N-... -

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Basic information

Entry
Database: PDB / ID: 8qz7
TitleStructure of human ceramide synthase 6 (CerS6) in complex with N-palmitoyl fumonisin B1
Components
  • Isoform 2 of Ceramide synthase 6
  • Nanobody-22
KeywordsMEMBRANE PROTEIN / CERAMIDE / SPHINGOLIPID / MYCOTOXIN / FUMONISIN B1 / INHIBITORY PRODUCT
Function / homology
Function and homology information


sphingoid base N-palmitoyltransferase / sphingosine N-acyltransferase activity / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / ceramide biosynthetic process / 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
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / : / Ceramide synthase 6
Similarity search - Component
Biological speciesHomo sapiens (human)
Vicugna pacos (alpaca)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3 Å
AuthorsPascoa, T.C. / Pike, A.C.W. / Chi, G. / Stefanic, S. / Quigley, A. / Chalk, R. / Mukhopadhyay, S.M.M. / Venkaya, S. / Dix, C. / Moreira, T. ...Pascoa, T.C. / Pike, A.C.W. / Chi, G. / Stefanic, S. / Quigley, A. / Chalk, R. / Mukhopadhyay, S.M.M. / Venkaya, S. / Dix, C. / Moreira, T. / Tessitore, A. / Cole, V. / Chu, A. / Elkins, J.M. / Pautsch, A. / Schnapp, G. / Carpenter, E.P. / Sauer, D.B.
Funding support United Kingdom, 2items
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, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update
Revision 1.2Mar 26, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Isoform 2 of Ceramide synthase 6
B: Nanobody-22
D: Nanobody-22
C: Isoform 2 of Ceramide synthase 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)118,92110
Polymers114,9784
Non-polymers3,9436
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "A" and (resid 2 through 35 or resid 37 through 403))
d_2ens_1(chain "C" and (resid 2 through 35 or resid 37 through 403))
d_1ens_2chain "B"
d_2ens_2chain "D"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg auth comp-IDBeg label comp-IDEnd auth comp-IDEnd label comp-IDAuth asym-IDLabel asym-IDAuth seq-IDLabel seq-ID
d_11ens_1ALAALAALAALAAA2 - 352 - 35
d_12ens_1ASPASPLYSLYSAA37 - 33437 - 334
d_13ens_1XBXXBXXBXXBXAE401
d_14ens_1PC1PC1PC1PC1AF402
d_15ens_1NAGNAGNAGNAGAG403
d_21ens_1ALAALAALAALACD2 - 352 - 35
d_22ens_1ASPASPLYSLYSCD37 - 33437 - 334
d_23ens_1XBXXBXXBXXBXCH401
d_24ens_1PC1PC1PC1PC1CI402
d_25ens_1NAGNAGNAGNAGCJ403
d_11ens_2GLNGLNVALVALBB1 - 1241 - 124
d_21ens_2GLNGLNVALVALDC1 - 1241 - 124

NCS ensembles :
ID
ens_1
ens_2

NCS oper:
IDCodeMatrixVector
1given(-0.999996949237, 0.00210758862964, 0.00128824971076), (-0.00211051731084, -0.999995182158, -0.00227626407974), (0.00128344607589, -0.00227897600871, 0.999996579511)235.796378736, 236.704622512, 0.0635536008364
2given(-0.999995807144, 0.00161654511785, 0.00240259777353), (-0.00162712100898, -0.999988967821, -0.00440643996883), (0.00239544805862, -0.00441033081058, 0.999987405326)235.70057889, 236.940565096, 0.0808215080208

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Components

#1: Protein Isoform 2 of Ceramide synthase 6 / CerS6 / LAG1 longevity assurance homolog 6 / Sphingoid base N-palmitoyltransferase CERS6


Mass: 42418.348 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CERS6, LASS6 / Plasmid: pHTBV1.1-CT10H-SIII-LIC / Production host: Homo sapiens (human)
References: UniProt: Q6ZMG9, sphingoid base N-palmitoyltransferase
#2: Antibody Nanobody-22


Mass: 15070.657 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Vicugna pacos (alpaca) / Plasmid: pDX / Production host: Escherichia coli (E. coli) / Strain (production host): WK6
#3: Chemical ChemComp-XBX / (2~{R})-2-[2-[(5~{R},6~{R},7~{S},9~{S},11~{R},16~{R},18~{S},19~{S})-6-[(3~{R})-3-carboxy-5-oxidanyl-5-oxidanylidene-pentanoyl]oxy-19-(hexadecanoylamino)-5,9-dimethyl-11,16,18-tris(oxidanyl)icosan-7-yl]oxy-2-oxidanylidene-ethyl]butanedioic acid


Mass: 960.239 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C50H89NO16 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE


Mass: 790.145 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
Has ligand of interestY
Has protein modificationY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: CerS6-Nb22 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightValue: 0.1148 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293F GnTI- / Plasmid: pHTBV1.1-CT10H-SIII-LIC
Buffer solutionpH: 7.5
Details: 20 mM HEPES pH 7.5, 200 mM NaCl, and 0.01 % (w/v) GDN
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2200 mMsodium chloride1
30.01 % (w/v)glyco-diosgenin1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: SEC-purified
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 1.34 sec. / Electron dose: 56.36 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 18386

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.1particle selection
2EPUimage acquisition
4cryoSPARC3.3.1CTF correctionPatch CTF
5RELION3.1.3CTF correctionPer-particle CTF refinement
8UCSF ChimeraX1.4model fitting
9Coot0.9.8model fitting
11PHENIX1.20.1-4487model refinement
12cryoSPARC3.3.1initial Euler assignment
13cryoSPARC3.3.1final Euler assignmentnon-uniform refinement
14RELION3.1.3classification
15cryoSPARC3.3.13D reconstructionnon-uniform refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3998935
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 154239 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 83.23 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00277594
ELECTRON MICROSCOPYf_angle_d0.424110356
ELECTRON MICROSCOPYf_chiral_restr0.03821140
ELECTRON MICROSCOPYf_plane_restr0.00351268
ELECTRON MICROSCOPYf_dihedral_angle_d11.52982582
Refine LS restraints NCS
Ens-IDDom-IDAsym-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AAELECTRON MICROSCOPYNCS constraints9.620470537E-14
ens_2d_2BBELECTRON MICROSCOPYNCS constraints2.75783453332E-13

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