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- EMDB-21369: Cryo-EM structure of the wild-type human serotonin transporter co... -

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Basic information

Entry
Database: EMDB / ID: EMD-21369
TitleCryo-EM structure of the wild-type human serotonin transporter complexed with Br-paroxetine and 8B6 Fab
Map datasharpened map
Sample
  • Complex: serotonin transporter 8B6 Fab complex
    • Complex: serotonin transporter
      • Protein or peptide: Sodium-dependent serotonin transporter
    • Complex: 8B6 antibody
      • Protein or peptide: 8B6 antibody, light chain
      • Protein or peptide: 8B6 antibody, heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: Br-paroxetine
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. / 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 subtype / Immunoglobulin / Immunoglobulin/major histocompatibility complex, conserved site / 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
MAb 6H10 light chain / 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 / Navratna V / Yang D
Funding support United States, 2 items
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, 2020-
Header (metadata) releaseMar 4, 2020-
Map releaseMar 11, 2020-
UpdateNov 25, 2020-
Current statusNov 25, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6vrk
  • Surface level: 6.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21369.png

Downloads & links

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Map

FileDownload / File: emd_21369.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map
Voxel sizeX=Y=Z: 0.648 Å
Density
Contour LevelBy AUTHOR: 6.5 / Movie #1: 6.5
Minimum - Maximum-21.074888 - 26.36943
Average (Standard dev.)-0.0010400241 (±0.9747236)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 259.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.6480.6480.648
M x/y/z400400400
origin x/y/z0.0000.0000.000
length x/y/z259.200259.200259.200
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ400400400
MAP C/R/S321
start NC/NR/NS000
NC/NR/NS400400400
D min/max/mean-21.07526.369-0.001

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Supplemental data

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Additional map: unsharpened map

Fileemd_21369_additional.map
Annotationunsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap2

Fileemd_21369_half_map_1.map
Annotationhalfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: halfmap1

Fileemd_21369_half_map_2.map
Annotationhalfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : serotonin transporter 8B6 Fab complex

EntireName: serotonin transporter 8B6 Fab complex
Components
  • Complex: serotonin transporter 8B6 Fab complex
    • Complex: serotonin transporter
      • Protein or peptide: Sodium-dependent serotonin transporter
    • Complex: 8B6 antibody
      • Protein or peptide: 8B6 antibody, light chain
      • Protein or peptide: 8B6 antibody, heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: DODECYL-BETA-D-MALTOSIDE
  • Ligand: Br-paroxetine

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Supramolecule #1: serotonin transporter 8B6 Fab complex

SupramoleculeName: serotonin transporter 8B6 Fab complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Molecular weightTheoretical: 105 KDa

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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: 8B6 antibody

SupramoleculeName: 8B6 antibody / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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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: 70.371305 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: METTPLNSQK QLSACEDGED CQENGVLQKV VPTPGDKVES GQISNGYSAV PSPGAGDDTR HSIPATTTTL VAELHQGERE TWGKKVDFL LSVIGYAVDL GNVWRFPYIC YQNGGGAFLL PYTIMAIFGG IPLFYMELAL GQYHRNGCIS IWRKICPIFK G IGYAICII ...String:
METTPLNSQK QLSACEDGED CQENGVLQKV VPTPGDKVES GQISNGYSAV PSPGAGDDTR HSIPATTTTL VAELHQGERE TWGKKVDFL LSVIGYAVDL GNVWRFPYIC YQNGGGAFLL PYTIMAIFGG IPLFYMELAL GQYHRNGCIS IWRKICPIFK G IGYAICII AFYIASYYNT IMAWALYYLI SSFTDQLPWT SCKNSWNTGN CTNYFSEDNI TWTLHSTSPA EEFYTRHVLQ IH RSKGLQD LGGISWQLAL CIMLIFTVIY FSIWKGVKTS GKVVWVTATF PYIILSVLLV RGATLPGAWR GVLFYLKPNW QKL LETGVW IDAAAQIFFS LGPGFGVLLA FASYNKFNNN CYQDALVTSV VNCMTSFVSG FVIFTVLGYM AEMRNEDVSE VAKD AGPSL LFITYAEAIA NMPASTFFAI IFFLMLITLG LDSTFAGLEG VITAVLDEFP HVWAKRRERF VLAVVITCFF GSLVT LTFG GAYVVKLLEE YATGPAVLTV ALIEAVAVSW FYGITQFCRD VKEMLGFSPG WFWRICWVAI SPLFLLFIIC SFLMSP PQL RLFQYNYPYW SIILGYCIGT SSFICIPTYI AYRLIITPGT FKERIIKSIT PETPTEIPCG DIRLNAV

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Macromolecule #2: 8B6 antibody, light chain

MacromoleculeName: 8B6 antibody, light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.718217 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: DIVMTQSHKF MSTSVGDRVS ITCKASQDVS TAVAWYQQKP GQSPKLLIYS ASYRYTGVPD RFTGSGSGTD FTFTISSVQA EDLAVYYCQ QHYSIPRTFG GGTKLEIKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS ...String:
DIVMTQSHKF MSTSVGDRVS ITCKASQDVS TAVAWYQQKP GQSPKLLIYS ASYRYTGVPD RFTGSGSGTD FTFTISSVQA EDLAVYYCQ QHYSIPRTFG GGTKLEIKRA DAAPTVSIFP PSSEQLTSGG ASVVCFLNNF YPKDINVKWK IDGSERQNGV L NSWTDQDS KDSTYSMSST LTLTKDEYER HNSYTCEATH KTSTSPIVKS FNRNEC

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Macromolecule #3: 8B6 antibody, heavy chain

MacromoleculeName: 8B6 antibody, heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.688365 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EVQLQQSGPE LVKPGASVKI SCKASGYTFT DYYMNWVKQS HGKSLEWIGN INPNNGGTSY NQKFKGKATL TVDKSSTTAY MELRSLTSE DSAVYYCTRS PVRPYYFDYW GQGTTLTVSS AKTTPPSVYP LAPGCGDTTG SSVTLGCLVK GYFPESVTVT W NSGSLSSS ...String:
EVQLQQSGPE LVKPGASVKI SCKASGYTFT DYYMNWVKQS HGKSLEWIGN INPNNGGTSY NQKFKGKATL TVDKSSTTAY MELRSLTSE DSAVYYCTRS PVRPYYFDYW GQGTTLTVSS AKTTPPSVYP LAPGCGDTTG SSVTLGCLVK GYFPESVTVT W NSGSLSSS VHTFPALLQS GLYTMSSSVT VPSSTWPSQT VTCSVAHPAS STTVDKKLEP SGP

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Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #5: DODECYL-BETA-D-MALTOSIDE

MacromoleculeName: DODECYL-BETA-D-MALTOSIDE / type: ligand / ID: 5 / 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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Macromolecule #6: Br-paroxetine

MacromoleculeName: Br-paroxetine / type: ligand / ID: 6 / Number of copies: 1 / Formula: RFS
Molecular weightTheoretical: 390.271 Da
Chemical component information

ChemComp-RFS:
Br-paroxetine

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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

Concentration4 mg/mL
BufferpH: 8
Component:
ConcentrationName
20.0 mMTris
100.0 mMsodium chloride
1.0 mMDodecyl maltopyranoside
0.2 mMCholesteryl hemisuccinate
GridDetails: unspecified
VitrificationCryogen name: ETHANE
Detailsmonodisperse

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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 K3 BIOQUANTUM (6k x 4k) / Average electron dose: 54.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: CTFFIND
Startup modelType of model: OTHER / Details: ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cisTEM
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 503993
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6awn

RefinementProtocol: RIGID BODY FIT
Output model

PDB-6vrk:
Cryo-EM structure of the wild-type human serotonin transporter complexed with Br-paroxetine and 8B6 Fab

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