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- PDB-6vrk: Cryo-EM structure of the wild-type human serotonin transporter co... -

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Basic information

Entry
Database: PDB / ID: 6vrk
TitleCryo-EM structure of the wild-type human serotonin transporter complexed with Br-paroxetine and 8B6 Fab
Components
  • (8B6 antibody, ...) x 2
  • Sodium-dependent serotonin transporter
KeywordsTRANSPORT PROTEIN / antidepressant / complex / transporter / antibody
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 / negative regulation of synaptic transmission, dopaminergic / sperm ejaculation / neurotransmitter transmembrane transporter activity ...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 / negative regulation of synaptic transmission, dopaminergic / sperm ejaculation / neurotransmitter transmembrane transporter activity / enteric nervous system development / serotonin uptake / cellular response to cGMP / negative regulation of organ growth / sodium ion binding / monoamine transmembrane transporter activity / monoamine transport / conditioned place preference / serotonin binding / vasoconstriction / brain morphogenesis / neurotransmitter transport / antiporter activity / syntaxin-1 binding / amino acid transport / nitric-oxide synthase binding / membrane depolarization / social behavior / behavioral response to cocaine / negative regulation of neuron differentiation / sodium ion transmembrane transport / endomembrane system / monoatomic cation channel activity / positive regulation of cell cycle / cellular response to retinoic acid / response to nutrient / response to toxic substance / memory / platelet aggregation / circadian rhythm / actin filament binding / integrin binding / response to estradiol / presynaptic membrane / postsynaptic membrane / endosome membrane / response to hypoxia / response to xenobiotic stimulus / neuron projection / immune response / membrane raft / focal adhesion / synapse / positive regulation of gene expression / extracellular space / 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. / Immunoglobulin V-Type / Immunoglobulin V-set domain ...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. / Immunoglobulin V-Type / Immunoglobulin V-set domain / Immunoglobulin V-set domain / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulin subtype / Immunoglobulin / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Br-paroxetine / MAb 6H10 light chain / Sodium-dependent serotonin transporter
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsColeman, J.A. / Navratna, V. / Yang, D.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Mental Health (NIH/NIMH)5R37MH070039 United States
Howard Hughes Medical Institute (HHMI) United States
CitationJournal: Elife / Year: 2020
Title: Chemical and structural investigation of the paroxetine-human serotonin transporter complex.
Authors: Jonathan A Coleman / Vikas Navratna / Daniele Antermite / Dongxue Yang / James A Bull / Eric Gouaux /
Abstract: Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally ...Antidepressants target the serotonin transporter (SERT) by inhibiting serotonin reuptake. Structural and biochemical studies aiming to understand binding of small-molecules to conformationally dynamic transporters like SERT often require thermostabilizing mutations and antibodies to stabilize a specific conformation, leading to questions about relationships of these structures to the bonafide conformation and inhibitor binding poses of wild-type transporter. To address these concerns, we determined the structures of ∆N72/∆C13 and ts2-inactive SERT bound to paroxetine analogues using single-particle cryo-EM and x-ray crystallography, respectively. We synthesized enantiopure analogues of paroxetine containing either bromine or iodine instead of fluorine. We exploited the anomalous scattering of bromine and iodine to define the pose of these inhibitors and investigated inhibitor binding to Asn177 mutants of ts2-active SERT. These studies provide mutually consistent insights into how paroxetine and its analogues bind to the central substrate-binding site of SERT, stabilize the outward-open conformation, and inhibit serotonin transport.
History
DepositionFeb 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_site / struct_site_gen
Item: _chem_comp.name / _entity.pdbx_description ..._chem_comp.name / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.pdbx_role
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 1.2Sep 23, 2020Group: Database references / Structure summary / Category: chem_comp / citation / citation_author
Item: _chem_comp.pdbx_synonyms / _citation.country ..._chem_comp.pdbx_synonyms / _citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

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Assembly

Deposited unit
A: Sodium-dependent serotonin transporter
C: 8B6 antibody, light chain
B: 8B6 antibody, heavy chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)119,1217
Polymers117,7783
Non-polymers1,3434
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5310 Å2
ΔGint-6 kcal/mol
Surface area32510 Å2

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Components

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Antibody , 2 types, 2 molecules CB

#2: Antibody 8B6 antibody, light chain


Mass: 23718.217 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: A0A0U5BC76
#3: Antibody 8B6 antibody, heavy chain


Mass: 23688.365 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Production host: Spodoptera frugiperda (fall armyworm)

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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: 70371.305 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC6A4, HTT, SERT / Cell line (production host): HEK293 GnTi- / Production host: Homo sapiens (human) / References: UniProt: P31645
#6: Chemical ChemComp-RFS / Br-paroxetine / (3S,4R)-3-{[(2H-1,3-benzodioxol-5-yl)oxy]methyl}-4-(4-bromophenyl)piperidine


Mass: 390.271 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C19H20BrNO3 / Feature type: SUBJECT OF INVESTIGATION

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Sugars , 2 types, 3 molecules

#4: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#5: 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 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

Component
IDNameTypeEntity IDParent-IDSource
1serotonin transporter 8B6 Fab complexCOMPLEX#1-#30MULTIPLE SOURCES
2serotonin transporterCOMPLEX#11RECOMBINANT
38B6 antibodyCOMPLEX#2-#31RECOMBINANT
Molecular weightValue: 0.105 MDa / Experimental value: NO
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
23Spodoptera frugiperda (fall armyworm)7108
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
120 mMTris1
2100 mMsodium chloride1
31 mMDodecyl maltopyranoside1
40.2 mMCholesteryl hemisuccinate1
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: monodisperse
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: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameCategory
1DoG Pickerparticle selection
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cisTEMfinal Euler assignment
12RELIONclassification
CTF correctionType: PHASE FLIPPING ONLY
3D reconstructionResolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 503993 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 6AWN

6awn

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0026327
ELECTRON MICROSCOPYf_angle_d0.5388621
ELECTRON MICROSCOPYf_dihedral_angle_d9.8233584
ELECTRON MICROSCOPYf_chiral_restr0.037966
ELECTRON MICROSCOPYf_plane_restr0.0041051

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