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- PDB-8j74: Human high-affinity choline transporter CHT1 in the HC-3-bound ou... -

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Basic information

Entry
Database: PDB / ID: 8j74
TitleHuman high-affinity choline transporter CHT1 in the HC-3-bound outward-facing open conformation, dimeric state
ComponentsHigh affinity choline transporter 1
KeywordsMEMBRANE PROTEIN / CHT1 / SLC5A7 / high affinity choline transporter / 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 / Transport of bile salts and organic acids, metal ions and amine compounds / choline transmembrane transporter activity / Acetylcholine Neurotransmitter Release Cycle / neuromuscular synaptic transmission / choline transport / choline binding ...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 / neuromuscular synaptic transmission / choline transport / choline binding / 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
Lauryl Maltose Neopentyl Glycol / Chem-HC6 / HEXADECANE / CHOLESTEROL HEMISUCCINATE / High affinity choline transporter 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsGao, Y. / Qiu, Y. / Zhao, Y.
Funding support China, 1items
OrganizationGrant numberCountry
Chinese Academy of SciencesXDB37030304 China
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Transport mechanism of presynaptic high-affinity choline uptake by CHT1.
Authors: Yunlong Qiu / Yiwei Gao / Bo Huang / Qinru Bai / Yan Zhao /
Abstract: Choline is a vital nutrient and a precursor for the biosynthesis of essential metabolites, including acetylcholine (ACh), that play a central role in fetal development, especially in the brain. In ...Choline is a vital nutrient and a precursor for the biosynthesis of essential metabolites, including acetylcholine (ACh), that play a central role in fetal development, especially in the brain. In cholinergic neurons, the high-affinity choline transporter (CHT1) provides an extraordinarily efficient reuptake mechanism to reutilize choline derived from intrasynaptical ACh hydrolysis and maintain ACh synthesis in the presynapse. Here, we determined structures of human CHT1 in three discrete states: the outward-facing state bound with the competitive inhibitor hemicholinium-3 (HC-3); the inward-facing occluded state bound with the substrate choline; and the inward-facing apo open state. Our structures and functional characterizations elucidate how the inhibitor and substrate are recognized. Moreover, our findings shed light on conformational changes when transitioning from an outward-facing to an inward-facing state and establish a framework for understanding the transport cycle, which relies on the stabilization of the outward-facing state by a short intracellular helix, IH1.
History
DepositionApr 27, 2023Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Apr 3, 2024Provider: repository / Type: Initial release
Revision 1.1Apr 10, 2024Group: Database references / Category: citation
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Apr 17, 2024Group: Database references / Category: citation_author
Revision 1.3Apr 24, 2024Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.4May 1, 2024Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.5Nov 6, 2024Group: Data collection / Structure summary
Category: em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: High affinity choline transporter 1
B: High affinity choline transporter 1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,91712
Polymers126,4782
Non-polymers5,43910
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein / Sugars , 2 types, 4 molecules AB

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


Mass: 63239.145 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC5A7, CHT1 / Cell line (production host): HEK 293-F / Production host: Homo sapiens (human) / References: UniProt: Q9GZV3
#2: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2 / Source method: obtained synthetically
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}LINUCSPDB-CARE

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Non-polymers , 4 types, 8 molecules

#3: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C31H50O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-AV0 / Lauryl Maltose Neopentyl Glycol / 2,2-didecylpropane-1,3-bis-b-D-maltopyranoside / 2-decyl-2-{[(4-O-alpha-D-glucopyranosyl-beta-D-glucopyranosyl)oxy]methyl}dodecyl4-O-alpha-D-glucopyranosyl-beta-D-glucopyranoside


Mass: 1005.188 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C47H88O22 / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-R16 / HEXADECANE


Mass: 226.441 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C16H34 / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical ChemComp-HC6 / (2S,2'S)-2,2'-biphenyl-4,4'-diylbis(2-hydroxy-4,4-dimethylmorpholin-4-ium)


Mass: 414.538 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H34N2O4 / Feature type: SUBJECT OF INVESTIGATION

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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

ComponentName: Human high-affinity choline transporter 1 / Type: COMPLEX / Entity ID: #1 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample was obtained from the monodispersed peak fractions of the size-exclusion chromatography.
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 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: 165000 X / Nominal defocus max: 2200 nm / Nominal defocus min: 1200 nm / Cs: 2.7 mm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.3.1particle selection
2SerialEM3.6image acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.15model fitting
9Coot0.9.6.2-premodel refinement
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
Particle selectionNum. of particles selected: 827001
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 12269 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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