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- EMDB-42496: Cholinephosphotransferase in complex with CDP-choline and phospha... -

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Basic information

Entry
Database: EMDB / ID: EMD-42496
TitleCholinephosphotransferase in complex with CDP-choline and phosphatidylcholine
Map dataMembrane protein enzyme in state 2
Sample
  • Complex: yCPT1
    • Complex: yCPT1
      • Protein or peptide: Cholinephosphotransferase 1
    • Complex: nanobody25
  • Ligand: MAGNESIUM ION
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: [2-CYTIDYLATE-O'-PHOSPHONYLOXYL]-ETHYL-TRIMETHYL-AMMONIUM
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSHOCHOLINE
  • Ligand: water
Keywordslipid metabolism / phospholipid synthesis / membrane protein enzyme / choline metabolism / MEMBRANE PROTEIN
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
Cholinephosphotransferase 1
Similarity search - Component
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Lama glama (llama)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsRoberts JR / Maeda S / Ohi MD
Funding support1 items
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, 2023-
Header (metadata) releaseOct 30, 2024-
Map releaseOct 30, 2024-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_42496.png

Downloads & links

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Map

FileDownload / File: emd_42496.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMembrane protein enzyme in state 2
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å		1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.9622473 - 3.5045614
Average (Standard dev.)0.000113824615 (±0.066504054)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map B

Fileemd_42496_half_map_1.map
AnnotationHalf Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map A

Fileemd_42496_half_map_2.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : yCPT1

EntireName: yCPT1
Components
  • Complex: yCPT1
    • Complex: yCPT1
      • Protein or peptide: Cholinephosphotransferase 1
    • Complex: nanobody25
  • Ligand: MAGNESIUM ION
  • Ligand: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: [2-CYTIDYLATE-O'-PHOSPHONYLOXYL]-ETHYL-TRIMETHYL-AMMONIUM
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSHOCHOLINE
  • Ligand: water

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Supramolecule #1: yCPT1

SupramoleculeName: yCPT1 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #2: yCPT1

SupramoleculeName: yCPT1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #3: nanobody25

SupramoleculeName: nanobody25 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Lama glama (llama)

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Macromolecule #1: Cholinephosphotransferase 1

MacromoleculeName: Cholinephosphotransferase 1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)
Molecular weightTheoretical: 44.147277 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: (ACE)GFFIPQSSL GNLKLYKYQS DDRSFLSNHV LRPFWRKFAT IFPLWMAPNL VTLLGFCFII FNVLTTLYYD PYFDQE SPR WTYFSYAIGL FLYQTFDACD GMHARRTGQQ GPLGELFDHC IDSINTTLSM IPVCSMTGMG YTYMTIFSQF AILCSFY LS TWEEYHTHKL ...String:
(ACE)GFFIPQSSL GNLKLYKYQS DDRSFLSNHV LRPFWRKFAT IFPLWMAPNL VTLLGFCFII FNVLTTLYYD PYFDQE SPR WTYFSYAIGL FLYQTFDACD GMHARRTGQQ GPLGELFDHC IDSINTTLSM IPVCSMTGMG YTYMTIFSQF AILCSFY LS TWEEYHTHKL YLAEFCGPVE GIIVLCISFI AVGIYGPQTI WHTKVAQFSW QDFVFDVETV HLMYAFCTGA LIFNIVTA H TNVVRYYESQ STKSATPSKT AENISKAVNG LLPFFAYFSS IFTLVLIQPS FISLALILSI GFSVAFVVGR MIIAHLTMQ PFPMVNFPFL IPTIQLVLYA FMVYVLDYQK GSIVSALVWM GLGLTLAIHG MFINDIIYDI TTFLDIYALS IK

UniProtKB: Cholinephosphotransferase 1

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 3 / Number of copies: 6 / Formula: PCW
Molecular weightTheoretical: 787.121 Da
Chemical component information

ChemComp-PCW:
1,2-DIOLEOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DOPC, phospholipid*YM

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Macromolecule #4: [2-CYTIDYLATE-O'-PHOSPHONYLOXYL]-ETHYL-TRIMETHYL-AMMONIUM

MacromoleculeName: [2-CYTIDYLATE-O'-PHOSPHONYLOXYL]-ETHYL-TRIMETHYL-AMMONIUM
type: ligand / ID: 4 / Number of copies: 2 / Formula: CDC
Molecular weightTheoretical: 488.324 Da
Chemical component information

ChemComp-CDC:
[2-CYTIDYLATE-O'-PHOSPHONYLOXYL]-ETHYL-TRIMETHYL-AMMONIUM

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Macromolecule #5: 1,2-DIACYL-SN-GLYCERO-3-PHOSHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSHOCHOLINE / type: ligand / ID: 5 / Number of copies: 2 / Formula: PCF
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PCF:
1,2-DIACYL-SN-GLYCERO-3-PHOSHOCHOLINE

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 9 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration1.0 mg/mL
BufferpH: 7.5
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 2990 / Average exposure time: 4.4 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.5 µm / Nominal magnification: 81000
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

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.2.1) / Number images used: 971309
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

7898


Chain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsInitial 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Cross-correlation coefficient
Output model

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

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