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- PDB-9bfk: Cryo-EM structure of human CHT1 in the ML352 bound state -

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Basic information

Entry
Database: PDB / ID: 9bfk
TitleCryo-EM structure of human CHT1 in the ML352 bound state
ComponentsHigh affinity choline transporter 1
KeywordsTRANSPORT PROTEIN / Choline transporter
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 / : / 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 / : / 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 / dendrite / synapse / 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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.85 Å
AuthorsXue, J. / Jiang, Y.
Funding support United States, 3items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release
Revision 1.0Nov 6, 2024Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Nov 6, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Nov 6, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.0Nov 6, 2024Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Nov 6, 2024Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Nov 6, 2024Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.0Nov 6, 2024Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Jan 15, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
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Revision 1.2Jun 4, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1Jun 4, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: High affinity choline transporter 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,9142
Polymers66,5271
Non-polymers3871
Water00
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

#1: Protein High affinity choline transporter 1 / hCHT1 / Hemicholinium-3-sensitive choline transporter / CHT / Solute carrier family 5 member 7


Mass: 66526.625 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC5A7, CHT1 / Production host: Homo sapiens (human) / References: UniProt: Q9GZV3
#2: Chemical ChemComp-A1AOW / 4-methoxy-3-[(1-methylpiperidin-4-yl)oxy]-N-{[3-(propan-2-yl)-1,2-oxazol-5-yl]methyl}benzamide


Mass: 387.473 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H29N3O4
Has ligand of interestY
Has protein modificationN

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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: human CHT1 in the ML352 bound state / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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: 1800 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 60 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.85 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 317376 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033939
ELECTRON MICROSCOPYf_angle_d0.6615381
ELECTRON MICROSCOPYf_dihedral_angle_d4.341537
ELECTRON MICROSCOPYf_chiral_restr0.042624
ELECTRON MICROSCOPYf_plane_restr0.005649

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