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- PDB-8urp: Cholinephosphotransferase in complex with CDP-choline and phospha... -

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Basic information

Entry
Database: PDB / ID: 8urp
TitleCholinephosphotransferase in complex with CDP-choline and phosphatidylcholine
ComponentsCholinephosphotransferase 1
KeywordsMEMBRANE PROTEIN / lipid metabolism / phospholipid synthesis / membrane protein enzyme / choline metabolism
Function / homology
Function and homology information


Synthesis of PC / Synthesis of PE / diacylglycerol cholinephosphotransferase / diacylglycerol cholinephosphotransferase activity / CDP-choline pathway / mitochondrial outer membrane / endoplasmic reticulum membrane / endoplasmic reticulum / Golgi apparatus / metal ion binding
Similarity search - Function
Choline/ethanolamine phosphotransferase / CDP-alcohol phosphatidyltransferase / CDP-alcohol phosphatidyltransferase, transmembrane domain / : / CDP-alcohol phosphatidyltransferase / CDP-alcohol phosphatidyltransferases signature.
Similarity search - Domain/homology
Chem-CDC / 1,2-DIACYL-SN-GLYCERO-3-PHOSHOCHOLINE / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Cholinephosphotransferase 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsRoberts, J.R. / Maeda, S. / Ohi, M.D.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: Nat Commun / Year: 2025
Title: Structural basis for catalysis and selectivity of phospholipid synthesis by eukaryotic choline-phosphotransferase.
Authors: Jacquelyn R Roberts / Yasuhiro Horibata / Frank E Kwarcinski / Vinson Lam / Ashleigh M Raczkowski / Akane Hubbard / Betsy White / Hiroyuki Sugimoto / Gregory G Tall / Melanie D Ohi / Shoji Maeda /
Abstract: Phospholipids are the most abundant component in lipid membranes and are essential for the structural and functional integrity of the cell. In eukaryotic cells, phospholipids are primarily ...Phospholipids are the most abundant component in lipid membranes and are essential for the structural and functional integrity of the cell. In eukaryotic cells, phospholipids are primarily synthesized de novo through the Kennedy pathway that involves multiple enzymatic processes. The terminal reaction is mediated by a group of cytidine-5'-diphosphate (CDP)-choline /CDP-ethanolamine-phosphotransferases (CPT/EPT) that use 1,2-diacylglycerol (DAG) and CDP-choline or CDP-ethanolamine to produce phosphatidylcholine (PC) or phosphatidylethanolamine (PE) that are the main phospholipids in eukaryotic cells. Here we present the structure of the yeast CPT1 in multiple substrate-bound states. Structural and functional analysis of these binding-sites reveal the critical residues for the DAG acyl-chain preference and the choline/ethanolamine selectivity. Additionally, we present the structure in complex with a potent inhibitor characterized in this study. The ensemble of structures allows us to propose the reaction mechanism for phospholipid biosynthesis by the family of CDP-alcohol phosphotransferases (CDP-APs).
History
DepositionOct 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 30, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 6, 2024Group: Data collection / Source and taxonomy / Category: em_admin / em_entity_assembly_recombinant
Item: _em_admin.last_update / _em_entity_assembly_recombinant.id
Revision 1.2Nov 20, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update
Revision 1.3Nov 27, 2024Group: Data collection / Data processing / Category: em_3d_reconstruction / em_admin
Item: _em_3d_reconstruction.resolution / _em_admin.last_update
Revision 1.4Jan 15, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / struct
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update / _em_admin.title / _struct.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Cholinephosphotransferase 1
B: Cholinephosphotransferase 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)95,55916
Polymers88,2952
Non-polymers7,26514
Water1629
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein Cholinephosphotransferase 1


Mass: 44147.277 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Saccharomyces cerevisiae (brewer's yeast)
Gene: CPT1 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P17898

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Non-polymers , 5 types, 23 molecules

#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg
#3: Chemical
ChemComp-PCW / 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / (Z,Z)-4-HYDROXY-N,N,N-TRIMETHYL-10-OXO-7-[(1-OXO-9-OCTADECENYL)OXY]-3,5,9-TRIOXA-4-PHOSPHAHEPTACOS-18-EN-1-AMINIUM-4-OXIDE


Mass: 787.121 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C44H85NO8P / Comment: DOPC, phospholipid*YM
#4: Chemical ChemComp-CDC / [2-CYTIDYLATE-O'-PHOSPHONYLOXYL]-ETHYL-TRIMETHYL-AMMONIUM


Mass: 488.324 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C14H26N4O11P2 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-PCF / 1,2-DIACYL-SN-GLYCERO-3-PHOSHOCHOLINE


Mass: 734.039 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: C40H80NO8P
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 9 / Source method: isolated from a natural source / Formula: H2O

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1yCPT1COMPLEX#10RECOMBINANT
2yCPT1COMPLEX#11RECOMBINANT
3nanobody25COMPLEX#11RECOMBINANT
Molecular weight
IDEntity assembly-IDUnitsExperimental value
11MEGADALTONSNO
22
33
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
21Saccharomyces cerevisiae (brewer's yeast)4932
32Saccharomyces cerevisiae (brewer's yeast)4932
43Lama glama (llama)9844
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDStrain
11Spodoptera frugiperda (fall armyworm)7108
22Spodoptera frugiperda (fall armyworm)7108
33Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)866768Rosetta
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 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: 81000 X / Nominal defocus max: 2400 nm / Nominal defocus min: 1500 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4.4 sec. / Electron dose: 60 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2990
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.2.1particle selectiontemplate picking
2SerialEMimage acquisition
4cryoSPARC4.2.1CTF correction
7PHENIX1.20.1-4487model fitting
9PHENIX1.20.1-4487model refinement
13cryoSPARC4.2.13D reconstruction
CTF correctionDetails: Patch CTF correction / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 971309 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Initial model fitting was done using a model superposition function into a 3D volume in Chimera and then Phenix.real_space_refine was used for flexible fitting.
Atomic model buildingAccession code: P17898 / Source name: AlphaFold / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026932
ELECTRON MICROSCOPYf_angle_d0.4429382
ELECTRON MICROSCOPYf_dihedral_angle_d13.4542494
ELECTRON MICROSCOPYf_chiral_restr0.0391010
ELECTRON MICROSCOPYf_plane_restr0.0041110

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