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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


positive regulation of oligodendrocyte apoptotic process / sphingoid base N-palmitoyltransferase / sphingosine N-acyltransferase activity / sphingolipid biosynthetic process / Sphingolipid de novo biosynthesis / oligodendrocyte development / 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: Nature Structural & Molecular Biology / Year: 2024
Title: Structural basis of the mechanism and inhibition of a human ceramide synthase
Authors: Pascoa, T.C. / Pike, A.C.W. / Tautermann, C. / Chi, G. / Traub, M. / Quigley, A. / Chalk, R. / Stefanic, S. / Thamm, S. / Pautsch, A. / Carpenter, E.P. / Schnapp, G. / Sauer, D.B.
History
DepositionOct 26, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 13, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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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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