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- PDB-8izd: Cryo-EM structure of the C26-CoA-bound Lac1-Lip1 complex -

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Basic information

Entry
Database: PDB / ID: 8izd
TitleCryo-EM structure of the C26-CoA-bound Lac1-Lip1 complex
Components
  • Ceramide synthase LAC1
  • Ceramide synthase subunit LIP1
KeywordsTRANSFERASE / Substrate / Complex
Function / homology
Function and homology information


very-long-chain ceramide synthase / acyl-CoA ceramide synthase complex / Sphingolipid de novo biosynthesis / sphingosine N-acyltransferase activity / ceramide biosynthetic process / nuclear periphery / nuclear envelope / endoplasmic reticulum membrane / endoplasmic reticulum
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.
Similarity search - Domain/homology
Chem-6PL / Hexacosanoyl-CoA / Ceramide synthase LAC1 / Ceramide synthase subunit LIP1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsXie, T. / Fang, Q. / Gong, X.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: EMBO J / Year: 2023
Title: Structure and mechanism of a eukaryotic ceramide synthase complex.
Authors: Tian Xie / Qi Fang / Zike Zhang / Yanfei Wang / Feitong Dong / Xin Gong /
Abstract: Ceramide synthases (CerS) catalyze ceramide formation via N-acylation of a sphingoid base with a fatty acyl-CoA and are attractive drug targets for treating numerous metabolic diseases and cancers. ...Ceramide synthases (CerS) catalyze ceramide formation via N-acylation of a sphingoid base with a fatty acyl-CoA and are attractive drug targets for treating numerous metabolic diseases and cancers. Here, we present the cryo-EM structure of a yeast CerS complex, consisting of a catalytic Lac1 subunit and a regulatory Lip1 subunit, in complex with C26-CoA substrate. The CerS holoenzyme exists as a dimer of Lac1-Lip1 heterodimers. Lac1 contains a hydrophilic reaction chamber and a hydrophobic tunnel for binding the CoA moiety and C26-acyl chain of C26-CoA, respectively. Lip1 interacts with both the transmembrane region and the last luminal loop of Lac1 to maintain the proper acyl chain binding tunnel. A lateral opening on Lac1 serves as a potential entrance for the sphingoid base substrate. Our findings provide a template for understanding the working mechanism of eukaryotic ceramide synthases and may facilitate the development of therapeutic CerS modulators.
History
DepositionApr 7, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Dec 13, 2023Provider: repository / Type: Initial release
Revision 1.1Dec 20, 2023Group: Database references / Category: citation / Item: _citation.journal_volume
Revision 1.2Oct 16, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Ceramide synthase LAC1
B: Ceramide synthase subunit LIP1
C: Ceramide synthase LAC1
D: Ceramide synthase subunit LIP1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)141,90712
Polymers135,0364
Non-polymers6,8718
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Ceramide synthase LAC1 / Very-long-chain ceramide synthase LAC1


Mass: 50289.082 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: LAC1, DGT1, YKL008C, YKL156 / Production host: Homo sapiens (human)
References: UniProt: P28496, very-long-chain ceramide synthase
#2: Protein Ceramide synthase subunit LIP1 / LAG1/LAC1-interacting protein 1


Mass: 17228.682 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Gene: LIP1, YMR298W / Production host: Homo sapiens (human) / References: UniProt: Q03579
#3: Chemical
ChemComp-6PL / (4S,7R)-4-HYDROXY-N,N,N-TRIMETHYL-9-OXO-7-[(PALMITOYLOXY)METHYL]-3,5,8-TRIOXA-4-PHOSPHAHEXACOSAN-1-AMINIUM 4-OXIDE / 1-PALMITOYL-2-STEAROYL-SN-GLYCERO-3-PHOSPHOCHOLINE


Mass: 763.100 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C42H85NO8P / Comment: phospholipid*YM
#4: Chemical ChemComp-9NY / Hexacosanoyl-CoA / C26:0 Coenzyme A


Mass: 1146.209 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C47H86N7O17P3S / Feature type: SUBJECT OF INVESTIGATION
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: Lac1-Lip1 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Source (natural)Organism: Saccharomyces cerevisiae (strain ATCC 204508 / S288c) (yeast)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: NONE
3D reconstructionResolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 179070 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0058030
ELECTRON MICROSCOPYf_angle_d1.14410874
ELECTRON MICROSCOPYf_dihedral_angle_d25.5651296
ELECTRON MICROSCOPYf_chiral_restr0.0811156
ELECTRON MICROSCOPYf_plane_restr0.0071298

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