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- EMDB-22024: hEAAT3-Asymmetric-2o1i-in20mM-Asp -

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Basic information

Entry
Database: EMDB / ID: EMD-22024
TitlehEAAT3-Asymmetric-2o1i-in20mM-Asp
Map datahEAAT3 asymmetric trimer with 2 outward-facing protomers and 1 inward-facing protomer in 20mM Asp
Sample
  • Complex: human Excitatory amino acid transporter 3 in 20mM Asp
    • Protein or peptide: human Excitatory amino acid transporter 3
Function / homology
Function and homology information


response to decreased oxygen levels / D-aspartate transmembrane transport / response to anesthetic / regulation of protein targeting to membrane / neurotransmitter receptor transport to plasma membrane / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / cysteine transmembrane transporter activity / cysteine transport ...response to decreased oxygen levels / D-aspartate transmembrane transport / response to anesthetic / regulation of protein targeting to membrane / neurotransmitter receptor transport to plasma membrane / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / cysteine transmembrane transporter activity / cysteine transport / monoatomic anion channel activity / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / cellular response to mercury ion / Transport of inorganic cations/anions and amino acids/oligopeptides / conditioned place preference / zinc ion transmembrane transport / L-glutamate transmembrane transport / retina layer formation / cellular response to bisphenol A / L-glutamate transmembrane transporter activity / L-aspartate transmembrane transport / D-aspartate import across plasma membrane / cellular response to ammonium ion / proximal dendrite / glutathione biosynthetic process / righting reflex / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / grooming behavior / intracellular glutamate homeostasis / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / apical dendrite / neurotransmitter transport / transepithelial transport / response to morphine / intracellular zinc ion homeostasis / glutamate receptor signaling pathway / cellular response to cocaine / chloride transmembrane transporter activity / motor neuron apoptotic process / blood vessel morphogenesis / motor behavior / glutamate binding / dopamine receptor signaling pathway / maintenance of blood-brain barrier / positive regulation of heart rate / adult behavior / dopamine metabolic process / postsynaptic modulation of chemical synaptic transmission / superoxide metabolic process / heart contraction / perisynaptic space / glial cell projection / transport across blood-brain barrier / cellular response to organic cyclic compound / asymmetric synapse / response to axon injury / behavioral fear response / synaptic cleft / axon terminus / monoatomic ion transport / chloride transmembrane transport / response to amphetamine / neurogenesis / dendritic shaft / locomotory behavior / long-term synaptic potentiation / cell periphery / synapse organization / regulation of protein phosphorylation / brain development / Schaffer collateral - CA1 synapse / memory / cytokine-mediated signaling pathway / recycling endosome membrane / presynapse / late endosome membrane / gene expression / cellular response to oxidative stress / perikaryon / chemical synaptic transmission / early endosome membrane / negative regulation of neuron apoptotic process / dendritic spine / response to xenobiotic stimulus / membrane raft / apical plasma membrane / axon / dendrite / neuronal cell body / cell surface / endoplasmic reticulum / extracellular exosome / membrane / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Sodium:dicarboxylate symporter family signature 2. / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter superfamily / Sodium:dicarboxylate symporter family / Sodium:dicarboxylate symporter family signature 1.
Similarity search - Domain/homology
Excitatory amino acid transporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.71 Å
AuthorsQiu B / Matthies D / Boudker O
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS064357 United States
CitationJournal: Sci Adv / Year: 2021
Title: Cryo-EM structures of excitatory amino acid transporter 3 visualize coupled substrate, sodium, and proton binding and transport.
Authors: Biao Qiu / Doreen Matthies / Eva Fortea / Zhiheng Yu / Olga Boudker /
Abstract: Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the ...Human excitatory amino acid transporter 3 (hEAAT3) mediates glutamate uptake in neurons, intestine, and kidney. Here, we report cryo-EM structures of hEAAT3 in several functional states where the transporter is empty, bound to coupled sodium ions only, or fully loaded with three sodium ions, a proton, and the substrate aspartate. The structures suggest that hEAAT3 operates by an elevator mechanism involving three functionally independent subunits. When the substrate-binding site is near the cytoplasm, it has a remarkably low affinity for the substrate, perhaps facilitating its release and allowing the rapid transport turnover. The mechanism of the coupled uptake of the sodium ions and the substrate is conserved across evolutionarily distant families and is augmented by coupling to protons in EAATs. The structures further suggest a mechanism by which a conserved glutamate residue mediates proton symport.
History
DepositionMay 21, 2020-
Header (metadata) releaseApr 28, 2021-
Map releaseApr 28, 2021-
UpdateApr 28, 2021-
Current statusApr 28, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.027
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.027
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22024.png

Downloads & links

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Map

FileDownload / File: emd_22024.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationhEAAT3 asymmetric trimer with 2 outward-facing protomers and 1 inward-facing protomer in 20mM Asp
Voxel sizeX=Y=Z: 1.096 Å
Density
Contour LevelBy AUTHOR: 0.025 / Movie #1: 0.027
Minimum - Maximum-0.0874803 - 0.15943585
Average (Standard dev.)-1.0526747e-05 (±0.0048697838)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 241.12 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0961.0961.096
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z241.120241.120241.120
α/β/γ90.00090.00090.000
start NX/NY/NZ278280246
NX/NY/NZ474891
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.0870.159-0.000

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Supplemental data

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Sample components

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Entire : human Excitatory amino acid transporter 3 in 20mM Asp

EntireName: human Excitatory amino acid transporter 3 in 20mM Asp
Components
  • Complex: human Excitatory amino acid transporter 3 in 20mM Asp
    • Protein or peptide: human Excitatory amino acid transporter 3

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Supramolecule #1: human Excitatory amino acid transporter 3 in 20mM Asp

SupramoleculeName: human Excitatory amino acid transporter 3 in 20mM Asp / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: human Excitatory amino acid transporter 3

MacromoleculeName: human Excitatory amino acid transporter 3 / type: protein_or_peptide / ID: 1
Details: The Glycine and Proline at the N terminal are the residues left after PreScission Protease treatment
Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPMGKPARKG CEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PL IISSMIT GVAALDSNVS GKIGLRAVVY YFCTTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVD AMLDLI RNMFPENLVQ ACFQQYKTKR ...String:
GPMGKPARKG CEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PL IISSMIT GVAALDSNVS GKIGLRAVVY YFCTTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVD AMLDLI RNMFPENLVQ ACFQQYKTKR EEVKPPSDPE MNMTEESFTA VMTTAISKNK TKEYKIVGMY SDGI NVLGL IVFCLVFGLV IGKMGEKGQI LVDFFNALSD ATMKIVQIIM CYMPLGILFL IAGKIIEVED WEIFR KLGL YMATVLTGLA IHSIVILPLI YFIVVRKNPF RFAMGMAQAL LTALMISSSS ATLPVTFRCA EENNQV DKR ITRFVLPVGA TINMDGTALY EAVAAVFIAQ LNDLDLGIGQ IITISITATS ASIGAAGVPQ AGLVTMV IV LSAVGLPAED VTLIIAVDWL LDRFRTMVNV LGDAFGTGIV EKLSKKELEQ MDVSSEVNIV NPFALEST I LDNEDSDTKK SYVNGGFAVD KSDTISFTQT SQF

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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

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris
200.0 mMNaClSodium chloridesodium chloride
1.0 mMC9H15O6PTCEP
0.086 mMC56H92O25Digitonin glyco diosgenin
20.0 mMC4H7NO4L-Aspartate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average exposure time: 10.0 sec. / Average electron dose: 67.6 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1533624
CTF correctionSoftware - Name: RELION (ver. 3.0)
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 8
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.71 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 168777
FSC plot (resolution estimation)

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