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- EMDB-8941: Cryo-EM structure of the ts2-inactive human serotonin transporter... -

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Basic information

Entry
Database: EMDB / ID: EMD-8941
TitleCryo-EM structure of the ts2-inactive human serotonin transporter in complex with paroxetine and 15B8 Fab and 8B6 ScFv
Map data
SampleHuman serotonin transporter in complex with Fab/Fv bound to paroxetine:
serotonin transporter / 8B6 ScFv / 15B8 Fab / Sodium-dependent serotonin transporter / 8B6 antibody single chain / (15B8 antibody ...) x 2 / (ligand) x 2
Function / homology
Function and homology information


Serotonin clearance from the synaptic cleft / negative regulation of cerebellar granule cell precursor proliferation / monoamine transmembrane transporter activity / regulation of thalamus size / serotonin uptake / serotonin:sodium symporter activity / positive regulation of serotonin secretion / serotonergic synapse / sperm ejaculation / negative regulation of synaptic transmission, dopaminergic ...Serotonin clearance from the synaptic cleft / negative regulation of cerebellar granule cell precursor proliferation / monoamine transmembrane transporter activity / regulation of thalamus size / serotonin uptake / serotonin:sodium symporter activity / positive regulation of serotonin secretion / serotonergic synapse / sperm ejaculation / negative regulation of synaptic transmission, dopaminergic / monoamine transport / cocaine binding / serotonin binding / vasoconstriction / cellular response to cGMP / negative regulation of organ growth / brain morphogenesis / neurotransmitter biosynthetic process / syntaxin-1 binding / myosin binding / social behavior / nitric-oxide synthase binding / endomembrane system / negative regulation of neuron differentiation / Rab GTPase binding / integral component of postsynaptic membrane / positive regulation of cell cycle / cellular response to retinoic acid / response to nutrient / response to toxic substance / memory / integral component of presynaptic membrane / circadian rhythm / actin filament binding / protein complex oligomerization / response to estradiol / protein homooligomerization / response to drug / endosome membrane / response to hypoxia / synapse / neuron projection / membrane raft / focal adhesion / positive regulation of gene expression / protein homodimerization activity / plasma membrane / metal ion binding
Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family profile. / Sodium:neurotransmitter symporter, serotonin, N-terminal / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter family signature 2. / Serotonin (5-HT) neurotransmitter transporter, N-terminus / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter family
Sodium-dependent serotonin transporter
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsColeman JA / Yang D / Gouaux E
CitationJournal: Nature / Year: 2019
Title: Serotonin transporter-ibogaine complexes illuminate mechanisms of inhibition and transport.
Authors: Jonathan A Coleman / Dongxue Yang / Zhiyu Zhao / Po-Chao Wen / Craig Yoshioka / Emad Tajkhorshid / Eric Gouaux /
Abstract: The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons. Major depression and anxiety ...The serotonin transporter (SERT) regulates neurotransmitter homeostasis through the sodium- and chloride-dependent recycling of serotonin into presynaptic neurons. Major depression and anxiety disorders are treated using selective serotonin reuptake inhibitors-small molecules that competitively block substrate binding and thereby prolong neurotransmitter action. The dopamine and noradrenaline transporters, together with SERT, are members of the neurotransmitter sodium symporter (NSS) family. The transport activities of NSSs can be inhibited or modulated by cocaine and amphetamines, and genetic variants of NSSs are associated with several neuropsychiatric disorders including attention deficit hyperactivity disorder, autism and bipolar disorder. Studies of bacterial NSS homologues-including LeuT-have shown how their transmembrane helices (TMs) undergo conformational changes during the transport cycle, exposing a central binding site to either side of the membrane. However, the conformational changes associated with transport in NSSs remain unknown. To elucidate structure-based mechanisms for transport in SERT we investigated its complexes with ibogaine, a hallucinogenic natural product with psychoactive and anti-addictive properties. Notably, ibogaine is a non-competitive inhibitor of transport but displays competitive binding towards selective serotonin reuptake inhibitors. Here we report cryo-electron microscopy structures of SERT-ibogaine complexes captured in outward-open, occluded and inward-open conformations. Ibogaine binds to the central binding site, and closure of the extracellular gate largely involves movements of TMs 1b and 6a. Opening of the intracellular gate involves a hinge-like movement of TM1a and the partial unwinding of TM5, which together create a permeation pathway that enables substrate and ion diffusion to the cytoplasm. These structures define the structural rearrangements that occur from the outward-open to inward-open conformations, and provide insight into the mechanism of neurotransmitter transport and ibogaine inhibition.
Validation ReportPDB-ID: 6dzw

SummaryFull reportAbout validation report
History
DepositionJul 5, 2018-
Header (metadata) releaseMar 13, 2019-
Map releaseApr 24, 2019-
UpdateJun 5, 2019-
Current statusJun 5, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6dzw
  • Surface level: 5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_8941.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.04 Å/pix.
x 240 pix.
= 250.56 Å
1.04 Å/pix.
x 240 pix.
= 250.56 Å
1.04 Å/pix.
x 240 pix.
= 250.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.044 Å
Density
Contour LevelBy AUTHOR: 5.0 / Movie #1: 5
Minimum - Maximum-20.285844999999998 - 29.558367000000001
Average (Standard dev.)-0.000000000001032 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 250.56001 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0441.0441.044
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z250.560250.560250.560
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-20.28629.558-0.000

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Supplemental data

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Mask #1

Fileemd_8941_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire Human serotonin transporter in complex with Fab/Fv bound to paroxetine

EntireName: Human serotonin transporter in complex with Fab/Fv bound to paroxetine
Number of components: 10

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Component #1: protein, Human serotonin transporter in complex with Fab/Fv bound...

ProteinName: Human serotonin transporter in complex with Fab/Fv bound to paroxetine
Recombinant expression: No

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Component #2: protein, serotonin transporter

ProteinName: serotonin transporter / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: protein, 8B6 ScFv

ProteinName: 8B6 ScFv / Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: protein, 15B8 Fab

ProteinName: 15B8 Fab / Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Mus musculus (house mouse)

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Component #5: protein, Sodium-dependent serotonin transporter

ProteinName: Sodium-dependent serotonin transporter / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 60.47943 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)

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Component #6: protein, 8B6 antibody single chain

ProteinName: 8B6 antibody single chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 26.220395 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #7: protein, 15B8 antibody heavy chain

ProteinName: 15B8 antibody heavy chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 12.980533 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Mus musculus (house mouse)

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Component #8: protein, 15B8 antibody light chain

ProteinName: 15B8 antibody light chain / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 11.776065 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Mus musculus (house mouse)

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Component #9: ligand, Paroxetine

LigandName: Paroxetine / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 0.329365 kDa

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Component #10: ligand, N-ACETYL-D-GLUCOSAMINE

LigandName: N-ACETYL-D-GLUCOSAMINEN-Acetylglucosamine / Number of Copies: 3 / Recombinant expression: No
MassTheoretical: 0.221208 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionpH: 8
Support filmunspecified
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 50 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Number of projections: 214537
3D reconstructionResolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Output model

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