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- PDB-8wgr: Cryo-EM structure of inward-open state human norepinephrine trans... -

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Basic information

Entry
Database: PDB / ID: 8wgr
TitleCryo-EM structure of inward-open state human norepinephrine transporter NET bound with antidepressant desipramine in KCl condition.
ComponentsSodium-dependent noradrenaline transporter
KeywordsMEMBRANE PROTEIN / Norepinephrine transporter / NET / SLC6A2 / norepinephrine / dopamine / neurotransmitter / desipramine / bupropion / antidepressant. / TRANSPORT PROTEIN
Function / homology
Function and homology information


neurotransmitter:sodium symporter activity / Defective SLC6A2 causes orthostatic intolerance (OI) / norepinephrine uptake / norepinephrine:sodium symporter activity / dopamine:sodium symporter activity / norepinephrine transport / neurotransmitter transmembrane transporter activity / monoamine transmembrane transporter activity / monoamine transport / Na+/Cl- dependent neurotransmitter transporters ...neurotransmitter:sodium symporter activity / Defective SLC6A2 causes orthostatic intolerance (OI) / norepinephrine uptake / norepinephrine:sodium symporter activity / dopamine:sodium symporter activity / norepinephrine transport / neurotransmitter transmembrane transporter activity / monoamine transmembrane transporter activity / monoamine transport / Na+/Cl- dependent neurotransmitter transporters / neurotransmitter transport / amino acid transport / response to pain / dopamine uptake involved in synaptic transmission / neuronal cell body membrane / beta-tubulin binding / alpha-tubulin binding / sodium ion transmembrane transport / neuron cellular homeostasis / presynaptic membrane / actin binding / chemical synaptic transmission / response to xenobiotic stimulus / axon / cell surface / membrane / metal ion binding / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter, noradrenaline / Sodium:neurotransmitter symporter family signature 2. / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family / Sodium:neurotransmitter symporter family profile.
Similarity search - Domain/homology
Chem-DSM / : / Sodium-dependent noradrenaline transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsTan, J. / Xiao, Y. / Kong, F. / Lei, J. / Yuan, Y. / Yan, C.
Funding support China, 1items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2020YFA0509301 China
CitationJournal: Nature / Year: 2024
Title: Molecular basis of human noradrenaline transporter reuptake and inhibition.
Authors: Jiaxin Tan / Yuan Xiao / Fang Kong / Xiaochun Zhang / Hanwen Xu / Angqi Zhu / Yiming Liu / Jianlin Lei / Boxue Tian / Yafei Yuan / Chuangye Yan /
Abstract: Noradrenaline, also known as norepinephrine, has a wide range of activities and effects on most brain cell types. Its reuptake from the synaptic cleft heavily relies on the noradrenaline transporter ...Noradrenaline, also known as norepinephrine, has a wide range of activities and effects on most brain cell types. Its reuptake from the synaptic cleft heavily relies on the noradrenaline transporter (NET) located in the presynaptic membrane. Here we report the cryo-electron microscopy (cryo-EM) structures of the human NET in both its apo state and when bound to substrates or antidepressant drugs, with resolutions ranging from 2.5 Å to 3.5 Å. The two substrates, noradrenaline and dopamine, display a similar binding mode within the central substrate binding site (S1) and within a newly identified extracellular allosteric site (S2). Four distinct antidepressants, namely, atomoxetine, desipramine, bupropion and escitalopram, occupy the S1 site to obstruct substrate transport in distinct conformations. Moreover, a potassium ion was observed within sodium-binding site 1 in the structure of the NET bound to desipramine under the KCl condition. Complemented by structural-guided biochemical analyses, our studies reveal the mechanism of substrate recognition, the alternating access of NET, and elucidate the mode of action of the four antidepressants.
History
DepositionSep 22, 2023Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jul 31, 2024Provider: repository / Type: Initial release
Revision 1.1Aug 7, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.pdbx_database_id_PubMed / _citation_author.name / _em_admin.last_update
Revision 1.2Sep 4, 2024Group: Data collection / Database references / Category: citation / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Sodium-dependent noradrenaline transporter
hetero molecules


Theoretical massNumber of molelcules
Total (without water)69,9945
Polymers69,3871
Non-polymers6074
Water61334
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Sodium-dependent noradrenaline transporter / Norepinephrine transporter / NET / Solute carrier family 6 member 2


Mass: 69386.547 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A2, NAT1, NET1, SLC6A5 / Production host: Homo sapiens (human) / References: UniProt: P23975
#2: Chemical ChemComp-DSM / 3-(10,11-DIHYDRO-5H-DIBENZO[B,F]AZEPIN-5-YL)-N-METHYLPROPAN-1-AMINE / Desipramine;Norpramin


Mass: 266.381 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C18H22N2 / Feature type: SUBJECT OF INVESTIGATION / Comment: antidepressant, inhibitor*YM
#3: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl
#4: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 34 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: NET / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.11 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
SpecimenConc.: 10 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen 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: 1400 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
13cryoSPARC3D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212071 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0034570
ELECTRON MICROSCOPYf_angle_d0.5466233
ELECTRON MICROSCOPYf_dihedral_angle_d4.659632
ELECTRON MICROSCOPYf_chiral_restr0.04691
ELECTRON MICROSCOPYf_plane_restr0.004754

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