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- EMDB-44499: Cryo-EM structure of human CHT1 in the ML352 bound state -

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Basic information

Entry
Database: EMDB / ID: EMD-44499
TitleCryo-EM structure of human CHT1 in the ML352 bound state
Map dataML352, full map
Sample
  • Organelle or cellular component: human CHT1 in the ML352 bound state
    • Protein or peptide: High affinity choline transporter 1
  • Ligand: 4-methoxy-3-[(1-methylpiperidin-4-yl)oxy]-N-{[3-(propan-2-yl)-1,2-oxazol-5-yl]methyl}benzamide
KeywordsCholine transporter / TRANSPORT PROTEIN
Function / homology
Function and homology information


choline:sodium symporter activity / Defective SLC5A7 causes distal hereditary motor neuronopathy 7A (HMN7A) / Defective SLC5A7 causes distal hereditary motor neuronopathy 7A (HMN7A) / acetylcholine biosynthetic process / Transport of bile salts and organic acids, metal ions and amine compounds / choline transmembrane transporter activity / Acetylcholine Neurotransmitter Release Cycle / choline transport / choline binding / neuromuscular synaptic transmission ...choline:sodium symporter activity / Defective SLC5A7 causes distal hereditary motor neuronopathy 7A (HMN7A) / Defective SLC5A7 causes distal hereditary motor neuronopathy 7A (HMN7A) / acetylcholine biosynthetic process / Transport of bile salts and organic acids, metal ions and amine compounds / choline transmembrane transporter activity / Acetylcholine Neurotransmitter Release Cycle / choline transport / choline binding / neuromuscular synaptic transmission / synaptic transmission, cholinergic / neurotransmitter transport / neuromuscular junction / transmembrane transport / synaptic vesicle membrane / presynaptic membrane / early endosome membrane / perikaryon / in utero embryonic development / axon / synapse / dendrite / membrane / plasma membrane
Similarity search - Function
: / Sodium/solute symporter / Sodium/glucose symporter superfamily / Sodium:solute symporter family / Sodium:solute symporter family profile.
Similarity search - Domain/homology
High affinity choline transporter 1
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsXue J / Jiang Y
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM140892 United States
Welch FoundationI-1578 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Cell Discov / Year: 2024
Title: Structural mechanisms of human sodium-coupled high-affinity choline transporter CHT1.
Authors: Jing Xue / Hongwen Chen / Yong Wang / Youxing Jiang /
Abstract: Mammalian sodium-coupled high-affinity choline transporter CHT1 uptakes choline in cholinergic neurons for acetylcholine synthesis and plays a critical role in cholinergic neurotransmission. Here, we ...Mammalian sodium-coupled high-affinity choline transporter CHT1 uptakes choline in cholinergic neurons for acetylcholine synthesis and plays a critical role in cholinergic neurotransmission. Here, we present the high-resolution cryo-EM structures of human CHT1 in apo, substrate- and ion-bound, hemicholinium-3-inhibited, and ML352-inhibited states. These structures represent three distinct conformational states, elucidating the structural basis of the CHT1-mediated choline uptake mechanism. Three ion-binding sites, two for Na and one for Cl, are unambiguously defined in the structures, demonstrating that both ions are indispensable cofactors for high-affinity choline-binding and are likely transported together with the substrate in a 2:1:1 stoichiometry. The two inhibitor-bound CHT1 structures reveal two distinct inhibitory mechanisms and provide a potential structural platform for designing therapeutic drugs to manipulate cholinergic neuron activity. Combined with the functional analysis, this study provides a comprehensive view of the structural mechanisms underlying substrate specificity, substrate/ion co-transport, and drug inhibition of a physiologically important symporter.
History
DepositionApr 18, 2024-
Header (metadata) releaseNov 6, 2024-
Map releaseNov 6, 2024-
UpdateJun 4, 2025-
Current statusJun 4, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44499.png

Downloads & links

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Map

FileDownload / File: emd_44499.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationML352, full map
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.74 Å/pix.
x 240 pix.
= 177.12 Å		0.74 Å/pix.
x 240 pix.
= 177.12 Å		0.74 Å/pix.
x 240 pix.
= 177.12 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.738 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-2.280751 - 3.413823
Average (Standard dev.)0.0021873217 (±0.09137376)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 177.12 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: half B

Fileemd_44499_half_map_1.map
Annotationhalf B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half A

Fileemd_44499_half_map_2.map
Annotationhalf 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 : human CHT1 in the ML352 bound state

EntireName: human CHT1 in the ML352 bound state
Components
  • Organelle or cellular component: human CHT1 in the ML352 bound state
    • Protein or peptide: High affinity choline transporter 1
  • Ligand: 4-methoxy-3-[(1-methylpiperidin-4-yl)oxy]-N-{[3-(propan-2-yl)-1,2-oxazol-5-yl]methyl}benzamide

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Supramolecule #1: human CHT1 in the ML352 bound state

SupramoleculeName: human CHT1 in the ML352 bound state / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: High affinity choline transporter 1

MacromoleculeName: High affinity choline transporter 1 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 66.526625 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAFHVEGLIA IIVFYLLILL VGIWAAWRTK NSGSAEERSE AIIVGGRDIG LLVGGFTMTA TWVGGGYING TAEAVYVPGY GLAWAQAPI GYSLSLILGG LFFAKPMRSK GYVTMLDPFQ QIYGKRMGGL LFIPALMGEM FWAAAIFSAL GATISVIIDV D MHISVIIS ...String:
MAFHVEGLIA IIVFYLLILL VGIWAAWRTK NSGSAEERSE AIIVGGRDIG LLVGGFTMTA TWVGGGYING TAEAVYVPGY GLAWAQAPI GYSLSLILGG LFFAKPMRSK GYVTMLDPFQ QIYGKRMGGL LFIPALMGEM FWAAAIFSAL GATISVIIDV D MHISVIIS ALIATLYTLV GGLYSVAYTD VVQLFCIFVG LWISVPFALS HPAVADIGFT AVHAKYQKPW LGTVDSSEVY SW LDSFLLL MLGGIPWQAY FQRVLSSSSA TYAQVLSFLA AFGCLVMAIP AILIGAIGAS TDWNQTAYGL PDPKTTEEAD MIL PIVLQY LCPVYISFFG LGAVSAAVMS SADSSILSAS SMFARNIYQL SFRQNASDKE IVWVMRITVF VFGASATAMA LLTK TVYGL WYLSSDLVYI VIFPQLLCVL FVKGTNTYGA VAGYVSGLFL RITGGEPYLY LQPLIFYPGY YPDDNGIYNQ KFPFK TLAM VTSFLTNICI SYLAKYLFES GTLPPKLDVF DAVVARHSEE NMDKTILVKN ENIKLDELAL VKPRQSMTLS STFTNK EAF LDVDSSPEGS GTEDNLQAAA GGSWSHPQFE KGGGSGGGSG GSAWSHPQFE K

UniProtKB: High affinity choline transporter 1

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Macromolecule #2: 4-methoxy-3-[(1-methylpiperidin-4-yl)oxy]-N-{[3-(propan-2-yl)-1,2...

MacromoleculeName: 4-methoxy-3-[(1-methylpiperidin-4-yl)oxy]-N-{[3-(propan-2-yl)-1,2-oxazol-5-yl]methyl}benzamide
type: ligand / ID: 2 / Number of copies: 1 / Formula: A1AOW
Molecular weightTheoretical: 387.473 Da

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k 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: 1.8 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 317376
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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