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- EMDB-8942: Cryo-EM structure of the ts2-active human serotonin transporter i... -

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Basic information

Entry
Database: EMDB / ID: EMD-8942
TitleCryo-EM structure of the ts2-active human serotonin transporter in complex with 15B8 Fab and 8B6 ScFv bound to ibogaine
Map datasharpened map
Sample
  • Complex: Human serotonin transporter in complex with Fab/Fv bound to ibogaine
    • Complex: serotonin transporter
      • Protein or peptide: Sodium-dependent serotonin transporter
    • Complex: 15B8 Fab
      • Protein or peptide: 15B8 antibody heavy chain
      • Protein or peptide: 15B8 antibody light chain
    • Complex: 8B6 ScFv
      • Protein or peptide: 8B6 antibody single chain
  • Ligand: (5beta)-12-methoxyibogamine
  • Ligand: DODECYL-BETA-D-MALTOSIDE
Function / homology
Function and homology information


negative regulation of cerebellar granule cell precursor proliferation / regulation of thalamus size / Serotonin clearance from the synaptic cleft / serotonergic synapse / positive regulation of serotonin secretion / cocaine binding / serotonin:sodium:chloride symporter activity / sperm ejaculation / neurotransmitter transmembrane transporter activity / negative regulation of synaptic transmission, dopaminergic ...negative regulation of cerebellar granule cell precursor proliferation / regulation of thalamus size / Serotonin clearance from the synaptic cleft / serotonergic synapse / positive regulation of serotonin secretion / cocaine binding / serotonin:sodium:chloride symporter activity / sperm ejaculation / neurotransmitter transmembrane transporter activity / negative regulation of synaptic transmission, dopaminergic / monoamine transmembrane transporter activity / serotonin uptake / monoamine transport / enteric nervous system development / negative regulation of organ growth / cellular response to cGMP / sodium ion binding / serotonin binding / neurotransmitter transport / vasoconstriction / brain morphogenesis / antiporter activity / syntaxin-1 binding / nitric-oxide synthase binding / social behavior / membrane depolarization / negative regulation of neuron differentiation / sodium ion transmembrane transport / endomembrane system / positive regulation of cell cycle / monoatomic cation channel activity / cellular response to retinoic acid / response to nutrient / memory / response to toxic substance / platelet aggregation / circadian rhythm / actin filament binding / integrin binding / response to estradiol / presynaptic membrane / postsynaptic membrane / response to hypoxia / endosome membrane / neuron projection / response to xenobiotic stimulus / membrane raft / focal adhesion / synapse / positive regulation of gene expression / identical protein binding / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter, serotonin, N-terminal / Serotonin (5-HT) neurotransmitter transporter, N-terminus / 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 serotonin transporter
Similarity search - Component
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsColeman JA / Yang D / Gouaux E
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)5R37MH070039 United States
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.
History
DepositionJul 5, 2018-
Header (metadata) releaseMar 13, 2019-
Map releaseApr 24, 2019-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6dzy
  • Surface level: 5.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8942.png

Downloads & links

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Map

FileDownload / File: emd_8942.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Voxel sizeX=Y=Z: 1.044 Å
Density
Contour LevelBy AUTHOR: 6.5 / Movie #1: 5.5
Minimum - Maximum-20.152925 - 32.55122
Average (Standard dev.)0.0031688476 (±1.3830576)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 313.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0441.0441.044
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z313.200313.200313.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-20.15332.5510.003

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Supplemental data

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Mask #1

Fileemd_8942_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: unsharpened map

Fileemd_8942_additional.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_8942_half_map_1.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_8942_half_map_2.map
Annotationhalf map 2
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram (log scale)

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Sample components

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

EntireName: Human serotonin transporter in complex with Fab/Fv bound to ibogaine
Components
  • Complex: Human serotonin transporter in complex with Fab/Fv bound to ibogaine
    • Complex: serotonin transporter
      • Protein or peptide: Sodium-dependent serotonin transporter
    • Complex: 15B8 Fab
      • Protein or peptide: 15B8 antibody heavy chain
      • Protein or peptide: 15B8 antibody light chain
    • Complex: 8B6 ScFv
      • Protein or peptide: 8B6 antibody single chain
  • Ligand: (5beta)-12-methoxyibogamine
  • Ligand: DODECYL-BETA-D-MALTOSIDE

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Supramolecule #1: Human serotonin transporter in complex with Fab/Fv bound to ibogaine

SupramoleculeName: Human serotonin transporter in complex with Fab/Fv bound to ibogaine
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #2-#4

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Supramolecule #2: serotonin transporter

SupramoleculeName: serotonin transporter / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Supramolecule #3: 15B8 Fab

SupramoleculeName: 15B8 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Mus musculus (house mouse)

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Supramolecule #4: 8B6 ScFv

SupramoleculeName: 8B6 ScFv / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #4
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #1: Sodium-dependent serotonin transporter

MacromoleculeName: Sodium-dependent serotonin transporter / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60.557508 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: RETWGKKVDF LLSVIGYAVD LGNVWRFPYI CYQNGGGAFL LPYTIMAIFG GIPLFYMELA LGQYHRNGCI SIWRKICPIF KGIGYAICI IAFYIASYYN TIMAWALYYL ISSFTDQLPW TSCKNSWNTG NCTNYFSEDN ITWTLHSTSP AEEFYTRHVL Q IHRSKGLQ ...String:
RETWGKKVDF LLSVIGYAVD LGNVWRFPYI CYQNGGGAFL LPYTIMAIFG GIPLFYMELA LGQYHRNGCI SIWRKICPIF KGIGYAICI IAFYIASYYN TIMAWALYYL ISSFTDQLPW TSCKNSWNTG NCTNYFSEDN ITWTLHSTSP AEEFYTRHVL Q IHRSKGLQ DLGGISWQLA LCIMLIFTVI YFSIWKGVKT SGKVVWVTAT FPYIALSVLL VRGATLPGAW RGVLFYLKPN WQ KLLETGV WIDAAAQIFF SLGPGFGVLL AFASYNKFNN NCYQDALVTS VVNCMTSFVS GFVIFTVLGY MAEMRNEDVS EVA KDAGPS LLFITYAEAI ANMPASTFFA IIFFLMLITL GLDSSFAGLE GVITAVLDEF PHVWAKRRER FVLAVVITCF FGSL VTLTF GGAYVVKLLE EYATGPAVLT VALIEAVAVS WFYGITQFCR DVKEMLGFSP GWFWRICWVA ISPLFLLFII ASFLM SPPQ LRLFQYNYPY WSIILGYAIG TSSFICIPTY IAYRLIITPG TFKERIIKSI TPE

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Macromolecule #2: 15B8 antibody heavy chain

MacromoleculeName: 15B8 antibody heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 12.980533 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
QVQLQQSGPE LVKLGASVRI SCKASGYRFS YSWMNWVKQR PGKGLEWIGR IYPGDGDTKY SGKFKGKATL TADKSSSTVY MQLSSLTSE DSAVYFCARS AYGSEGFAMD YWGQGTSVT

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Macromolecule #3: 15B8 antibody light chain

MacromoleculeName: 15B8 antibody light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 11.776065 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString:
DIVLTQSPAS LAVSLGQRAT ISCRASESVD NYGISFLNWF QQKPGQPPKL LIYAASNQGS GVPARFSGSG SGTYFSLNIH PMEEDDTAV YFCQQTKGVS WTFGGGTKVE I

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Macromolecule #4: 8B6 antibody single chain

MacromoleculeName: 8B6 antibody single chain / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.220395 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: EVQLQQSGPE LVKPGASVKI SCKASGYTFT DYYMNWVKQS HGKSLEWIGN INPNNGGTSY NQKFKGKATL TVDKSSTTAY MELRSLTSE DSAVYYCTRS PVRPYYFDYW GQGTTLTV(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK) ...String:
EVQLQQSGPE LVKPGASVKI SCKASGYTFT DYYMNWVKQS HGKSLEWIGN INPNNGGTSY NQKFKGKATL TVDKSSTTAY MELRSLTSE DSAVYYCTRS PVRPYYFDYW GQGTTLTV(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)DIVM TQSHKFMSTS VGDRVSITCK ASQDVSTAVA W YQQKPGQS PKLLIYSASY RYTGVPDRFT GSGSGTDFTF TISSVQAEDL AVYYCQQHYS IPRTFGGGTK LEI

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Macromolecule #6: (5beta)-12-methoxyibogamine

MacromoleculeName: (5beta)-12-methoxyibogamine / type: ligand / ID: 6 / Number of copies: 1 / Formula: HJM
Molecular weightTheoretical: 310.433 Da
Chemical component information

ChemComp-HJM:
(5beta)-12-methoxyibogamine / Ibogaine

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Macromolecule #7: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 7 / Number of copies: 1 / Formula: LMT
Molecular weightTheoretical: 510.615 Da
Chemical component information

ChemComp-LMT:
DODECYL-BETA-D-MALTOSIDE / detergent*YM

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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

BufferpH: 8
GridDetails: unspecified
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 60.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: Ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 153986
FSC plot (resolution estimation)

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