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- EMDB-37515: Cryo-EM structure of inward-open state human norepinephrine trans... -

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Basic information

Entry
Database: EMDB / ID: EMD-37515
TitleCryo-EM structure of inward-open state human norepinephrine transporter NET bound with antidepressant desipramine in KCl condition.
Map datacryoEM density map for hNET bhound with desipramine in KCl contidtion
Sample
  • Complex: NET
    • Protein or peptide: Sodium-dependent noradrenaline transporter
  • Ligand: 3-(10,11-DIHYDRO-5H-DIBENZO[B,F]AZEPIN-5-YL)-N-METHYLPROPAN-1-AMINE
  • Ligand: CHLORIDE ION
  • Ligand: POTASSIUM ION
  • Ligand: water
KeywordsNorepinephrine transporter / NET / SLC6A2 / norepinephrine / dopamine / neurotransmitter / desipramine / bupropion / antidepressant. / TRANSPORT PROTEIN / MEMBRANE 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
Sodium-dependent noradrenaline transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsTan J / Xiao Y / Kong F / Lei J / Yuan Y / Yan C
Funding support China, 1 items
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, 2023-
Header (metadata) releaseJul 31, 2024-
Map releaseJul 31, 2024-
UpdateSep 4, 2024-
Current statusSep 4, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_37515.png

Downloads & links

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Map

FileDownload / File: emd_37515.map.gz / Format: CCP4 / Size: 27 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationcryoEM density map for hNET bhound with desipramine in KCl contidtion
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 192 pix.
= 207.84 Å		1.08 Å/pix.
x 192 pix.
= 207.84 Å		1.08 Å/pix.
x 192 pix.
= 207.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.1
Minimum - Maximum-4.6025863 - 6.2467985
Average (Standard dev.)0.0019155975 (±0.15947683)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions192192192
Spacing192192192
CellA=B=C: 207.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_37515_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

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Half map: #2

Fileemd_37515_half_map_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: #1

Fileemd_37515_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : NET

EntireName: NET
Components
  • Complex: NET
    • Protein or peptide: Sodium-dependent noradrenaline transporter
  • Ligand: 3-(10,11-DIHYDRO-5H-DIBENZO[B,F]AZEPIN-5-YL)-N-METHYLPROPAN-1-AMINE
  • Ligand: CHLORIDE ION
  • Ligand: POTASSIUM ION
  • Ligand: water

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Supramolecule #1: NET

SupramoleculeName: NET / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 110 KDa

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Macromolecule #1: Sodium-dependent noradrenaline transporter

MacromoleculeName: Sodium-dependent noradrenaline transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 69.386547 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MLLARMNPQV QPENNGADTG PEQPLRARKT AELLVVKERN GVQCLLAPRD GDAQPRETWG KKIDFLLSVV GFAVDLANVW RFPYLCYKN GGGAFLIPYT LFLIIAGMPL FYMELALGQY NREGAATVWK ICPFFKGVGY AVILIALYVG FYYNVIIAWS L YYLFSSFT ...String:
MLLARMNPQV QPENNGADTG PEQPLRARKT AELLVVKERN GVQCLLAPRD GDAQPRETWG KKIDFLLSVV GFAVDLANVW RFPYLCYKN GGGAFLIPYT LFLIIAGMPL FYMELALGQY NREGAATVWK ICPFFKGVGY AVILIALYVG FYYNVIIAWS L YYLFSSFT LNLPWTDCGH TWNSPNCTDP KLLNGSVLGN HTKYSKYKFT PAAEFYERGV LHLHESSGIH DIGLPQWQLL LC LMVVVIV LYFSLWKGVK TSGKVVWITA TLPYFVLFVL LVHGVTLPGA SNGINAYLHI DFYRLKEATV WIDAATQIFF SLG AGFGVL IAFASYNKFD NNCYRDALLT SSINCITSFV SGFAIFSILG YMAHEHKVNI EDVATEGAGL VFILYPEAIS TLSG STFWA VVFFVMLLAL GLDSSMGGME AVITGLADDF QVLKRHRKLF TFGVTFSTFL LALFCITKGG IYVLTLLDTF AAGTS ILFA VLMEAIGVSW FYGVDRFSND IQQMMGFRPG LYWRLCWKFV SPAFLLFVVV VSIINFKPLT YDDYIFPPWA NWVGWG IAL SSMVLVPIYV IYKFLSTQGS LWERLAYGIT PENEHHLVAQ RDIRQFQLQH WLAI

UniProtKB: Sodium-dependent noradrenaline transporter

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Macromolecule #2: 3-(10,11-DIHYDRO-5H-DIBENZO[B,F]AZEPIN-5-YL)-N-METHYLPROPAN-1-AMINE

MacromoleculeName: 3-(10,11-DIHYDRO-5H-DIBENZO[B,F]AZEPIN-5-YL)-N-METHYLPROPAN-1-AMINE
type: ligand / ID: 2 / Number of copies: 2 / Formula: DSM
Molecular weightTheoretical: 266.381 Da
Chemical component information

ChemComp-DSM:
3-(10,11-DIHYDRO-5H-DIBENZO[B,F]AZEPIN-5-YL)-N-METHYLPROPAN-1-AMINE / antidepressant, inhibitor*YM

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Macromolecule #3: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 34 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration10 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.4000000000000001 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 212071
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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