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- EMDB-46969: Cholinephosphotransferase in complex with diacylglycerol in nanodisc -

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Basic information

Entry
Database: EMDB / ID: EMD-46969
TitleCholinephosphotransferase in complex with diacylglycerol in nanodisc
Map dataMembrane protein enzyme in nanodisc
Sample
  • Complex: Saccharomyces cerevisiae CDP-choline phosphotransferase in complex with diacyl-glycerol
    • Complex: nanobody24
    • Complex: yCPT1
      • Protein or peptide: Cholinephosphotransferase 1
Keywordslipid metabolism / phospholipid synthesis / membrane protein enzyme / choline metabolism / MEMBRANE PROTEIN
Biological speciesSaccharomyces cerevisiae (brewer's yeast) / Lama glama (llama) / Saccharomyces cerevisiae S288C (yeast)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.71 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
DepositionSep 10, 2024-
Header (metadata) releaseNov 13, 2024-
Map releaseNov 13, 2024-
UpdateJan 15, 2025-
Current statusJan 15, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46969.png

Downloads & links

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Map

FileDownload / File: emd_46969.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMembrane protein enzyme in nanodisc
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: 0.248
Minimum - Maximum-0.8627254 - 1.141223
Average (Standard dev.)-0.00032753174 (±0.022881752)
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 A

Fileemd_46969_half_map_1.map
AnnotationHalf Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_46969_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Saccharomyces cerevisiae CDP-choline phosphotransferase in comple...

EntireName: Saccharomyces cerevisiae CDP-choline phosphotransferase in complex with diacyl-glycerol
Components
  • Complex: Saccharomyces cerevisiae CDP-choline phosphotransferase in complex with diacyl-glycerol
    • Complex: nanobody24
    • Complex: yCPT1
      • Protein or peptide: Cholinephosphotransferase 1

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Supramolecule #1: Saccharomyces cerevisiae CDP-choline phosphotransferase in comple...

SupramoleculeName: Saccharomyces cerevisiae CDP-choline phosphotransferase in complex with diacyl-glycerol
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Supramolecule #2: nanobody24

SupramoleculeName: nanobody24 / type: complex / ID: 2 / Parent: 1
Source (natural)Organism: Lama glama (llama)

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Supramolecule #3: yCPT1

SupramoleculeName: yCPT1 / type: complex / ID: 3 / Parent: 1 / Macromolecule list: all
Source (natural)Organism: Saccharomyces cerevisiae (brewer's yeast)

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Macromolecule #1: Cholinephosphotransferase 1

MacromoleculeName: Cholinephosphotransferase 1 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Saccharomyces cerevisiae S288C (yeast)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGFFIPQSSL GNLKLYKYQS DDRSFLSNHV LRPFWRKFAT IFPLWMAPNL VTLLGFCFII FNVLTTLYY DPYFDQESPR WTYFSYAIGL FLYQTFDACD GMHARRTGQQ GPLGELFDHC I DSINTTLS MIPVCSMTGM GYTYMTIFSQ FAILCSFYLS TWEEYHTHKL ...String:
MGFFIPQSSL GNLKLYKYQS DDRSFLSNHV LRPFWRKFAT IFPLWMAPNL VTLLGFCFII FNVLTTLYY DPYFDQESPR WTYFSYAIGL FLYQTFDACD GMHARRTGQQ GPLGELFDHC I DSINTTLS MIPVCSMTGM GYTYMTIFSQ FAILCSFYLS TWEEYHTHKL YLAEFCGPVE GI IVLCISF IAVGIYGPQT IWHTKVAQFS WQDFVFDVET VHLMYAFCTG ALIFNIVTAH TNV VRYYES QSTKSATPSK TAENISKAVN GLLPFFAYFS SIFTLVLIQP SFISLALILS IGFS VAFVV GRMIIAHLTM QPFPMVNFPF LIPTIQLVLY AFMVYVLDYQ KGSIVSALVW MGLGL TLAI HGMFINDIIY DITTFLDIYA LSIKHPKEI

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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

Concentration2 mg/mL
BufferpH: 7.5
VitrificationCryogen name: NITROGEN

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / 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 / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.5 µm
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: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 72669
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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