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- PDB-6dzz: Cryo-EM Structure of the wild-type human serotonin transporter in... -

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Basic information

Entry
Database: PDB / ID: 6dzz
TitleCryo-EM Structure of the wild-type human serotonin transporter in complex with ibogaine and 15B8 Fab in the inward conformation
Components
  • (15B8 antibody ...) x 2
  • Sodium-dependent serotonin transporter
KeywordsTRANSPORT PROTEIN/IMMUNE SYSTEM / ibogaine / human serotonin transporter / Fab / TRANSPORT PROTEIN-IMMUNE SYSTEM complex
Function / homology
Function and homology information


negative regulation of cerebellar granule cell precursor proliferation / regulation of thalamus size / Serotonin clearance from the synaptic cleft / positive regulation of serotonin secretion / serotonergic synapse / cocaine binding / negative regulation of synaptic transmission, dopaminergic / serotonin:sodium:chloride symporter activity / cellular response to cGMP / enteric nervous system development ...negative regulation of cerebellar granule cell precursor proliferation / regulation of thalamus size / Serotonin clearance from the synaptic cleft / positive regulation of serotonin secretion / serotonergic synapse / cocaine binding / negative regulation of synaptic transmission, dopaminergic / serotonin:sodium:chloride symporter activity / cellular response to cGMP / enteric nervous system development / sodium ion binding / negative regulation of organ growth / neurotransmitter transmembrane transporter activity / serotonin uptake / monoamine transmembrane transporter activity / monoamine transport / serotonin binding / conditioned place preference / vasoconstriction / brain morphogenesis / syntaxin-1 binding / antiporter activity / neurotransmitter transport / nitric-oxide synthase binding / male mating behavior / amino acid transport / behavioral response to cocaine / membrane depolarization / negative regulation of neuron differentiation / monoatomic cation channel activity / cellular response to retinoic acid / positive regulation of cell cycle / response to nutrient / sodium ion transmembrane transport / endomembrane system / memory / platelet aggregation / response to toxic substance / circadian rhythm / actin filament binding / integrin binding / response to estradiol / presynaptic membrane / postsynaptic membrane / response to hypoxia / neuron projection / endosome membrane / membrane raft / response to xenobiotic stimulus / 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
(5beta)-12-methoxyibogamine / Sodium-dependent serotonin transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsYang, D. / Coleman, J.A. / Gouaux, E.
Funding support United States, 2items
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, 2018Deposition site: RCSB / Processing site: RCSB
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Structure visualization

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Assembly

Deposited unit
A: Sodium-dependent serotonin transporter
B: 15B8 antibody heavy chain
C: 15B8 antibody light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)87,0076
Polymers85,7623
Non-polymers1,2453
Water00
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4510 Å2
ΔGint-10 kcal/mol
Surface area35680 Å2

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Components

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Antibody , 2 types, 2 molecules BC

#2: Antibody 15B8 antibody heavy chain


Mass: 12980.533 Da / Num. of mol.: 1 / Fragment: Fab variable domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)
#3: Antibody 15B8 antibody light chain


Mass: 11776.065 Da / Num. of mol.: 1 / Fragment: Fab variable domain
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Mus musculus (house mouse)

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Protein / Non-polymers , 2 types, 2 molecules A

#1: Protein Sodium-dependent serotonin transporter / SERT / 5HT transporter / 5HTT / Solute carrier family 6 member 4


Mass: 61005.074 Da / Num. of mol.: 1 / Fragment: UNP residues 79-615
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A4, HTT, SERT / Production host: Homo sapiens (human) / References: UniProt: P31645
#5: Chemical ChemComp-HJM / (5beta)-12-methoxyibogamine


Mass: 310.433 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Formula: C20H26N2O / Comment: alkaloid*YM

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Sugars , 2 types, 2 molecules

#4: Polysaccharide 2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#6: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM

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Details

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

Component
IDNameTypeEntity IDParent-IDSource
1Human serotonin transporter in complex with Fab bound to ibogaineCOMPLEX#1-#30MULTIPLE SOURCES
2serotonin transporterCOMPLEX#11RECOMBINANT
315B8 FabCOMPLEX#2-#31RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
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
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.13_2998: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 368656 / Symmetry type: POINT
RefinementHighest resolution: 3.6 Å

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