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- EMDB-22021: hEAAT3-IFS-Apo -

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Basic information

Entry
Database: EMDB / ID: EMD-22021
TitlehEAAT3-IFS-Apo
Map datahEAAT3 inward-facing Apo state
Sample
  • Complex: inward facing hEAAT3 trimer Apo state
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: CHOLINE ION
KeywordshEAAT3 inward-facing Apo state / TRANSPORT PROTEIN
Function / homology
Function and homology information


D-aspartate transmembrane transport / regulation of protein targeting to membrane / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / response to anesthetic / cysteine transmembrane transporter activity / cysteine transport / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity ...D-aspartate transmembrane transport / regulation of protein targeting to membrane / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / response to anesthetic / cysteine transmembrane transporter activity / cysteine transport / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / neurotransmitter receptor transport to plasma membrane / response to decreased oxygen levels / cellular response to mercury ion / Transport of inorganic cations/anions and amino acids/oligopeptides / retina layer formation / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / zinc ion transmembrane transport / cellular response to bisphenol A / L-aspartate transmembrane transport / glutathione biosynthetic process / cellular response to ammonium ion / D-aspartate import across plasma membrane / righting reflex / apical dendrite / monoatomic anion channel activity / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / grooming behavior / intracellular glutamate homeostasis / Glutamate Neurotransmitter Release Cycle / proximal dendrite / L-glutamate import across plasma membrane / transepithelial transport / conditioned place preference / intracellular zinc ion homeostasis / cellular response to cocaine / blood vessel morphogenesis / motor behavior / neurotransmitter transport / response to morphine / motor neuron apoptotic process / chloride transmembrane transporter activity / glutamate binding / glutamate receptor signaling pathway / G protein-coupled dopamine receptor signaling pathway / positive regulation of heart rate / postsynaptic modulation of chemical synaptic transmission / maintenance of blood-brain barrier / superoxide metabolic process / heart contraction / adult behavior / perisynaptic space / dopamine metabolic process / cellular response to organic cyclic compound / glial cell projection / behavioral fear response / asymmetric synapse / transport across blood-brain barrier / response to axon injury / synaptic cleft / monoatomic ion transport / axon terminus / chloride transmembrane transport / response to amphetamine / neurogenesis / dendritic shaft / cell periphery / long-term synaptic potentiation / locomotory behavior / brain development / synapse organization / regulation of protein phosphorylation / Schaffer collateral - CA1 synapse / cytokine-mediated signaling pathway / memory / recycling endosome membrane / late endosome membrane / presynapse / cellular response to oxidative stress / gene expression / early endosome membrane / chemical synaptic transmission / perikaryon / negative regulation of neuron apoptotic process / dendritic spine / response to xenobiotic stimulus / membrane raft / apical plasma membrane / axon / neuronal cell body / dendrite / cell surface / endoplasmic reticulum / extracellular exosome / identical protein binding / membrane / 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: 3.03 Å
AuthorsQiu B / Matthies D
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) releaseMar 17, 2021-
Map releaseMar 17, 2021-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6x3f
  • Surface level: 0.019
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22021.png

Downloads & links

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Map

FileDownload / File: emd_22021.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationhEAAT3 inward-facing Apo state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 288 pix.
= 239.616 Å		0.83 Å/pix.
x 288 pix.
= 239.616 Å		0.83 Å/pix.
x 288 pix.
= 239.616 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.019 / Movie #1: 0.019
Minimum - Maximum-0.10791589 - 0.16292043
Average (Standard dev.)0.000014156628 (±0.0042244494)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 239.616 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z288288288
origin x/y/z0.0000.0000.000
length x/y/z239.616239.616239.616
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS288288288
D min/max/mean-0.1080.1630.000

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Supplemental data

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Sample components

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Entire : inward facing hEAAT3 trimer Apo state

EntireName: inward facing hEAAT3 trimer Apo state
Components
  • Complex: inward facing hEAAT3 trimer Apo state
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: CHOLINE ION

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Supramolecule #1: inward facing hEAAT3 trimer Apo state

SupramoleculeName: inward facing hEAAT3 trimer Apo state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Excitatory amino acid transporter 3

MacromoleculeName: 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
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.301168 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPMGKPARKG CEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PLIISSMITG VAALDSNVS GKIGLRAVVY YFCTTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVDAMLDLIR NMFPENLVQA C FQQYKTKR ...String:
GPMGKPARKG CEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PLIISSMITG VAALDSNVS GKIGLRAVVY YFCTTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVDAMLDLIR NMFPENLVQA C FQQYKTKR EEVKPPSDPE MNMTEESFTA VMTTAISKNK TKEYKIVGMY SDGINVLGLI VFCLVFGLVI GKMGEKGQIL VD FFNALSD ATMKIVQIIM CYMPLGILFL IAGKIIEVED WEIFRKLGLY MATVLTGLAI HSIVILPLIY FIVVRKNPFR FAM GMAQAL LTALMISSSS ATLPVTFRCA EENNQVDKRI TRFVLPVGAT INMDGTALYE AVAAVFIAQL NDLDLGIGQI ITIS ITATS ASIGAAGVPQ AGLVTMVIVL SAVGLPAEDV TLIIAVDWLL DRFRTMVNVL GDAFGTGIVE KLSKKELEQM DVSSE VNIV NPFALESTIL DNEDSDTKKS YVNGGFAVDK SDTISFTQTS QF

UniProtKB: Excitatory amino acid transporter 3

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Macromolecule #2: CHOLINE ION

MacromoleculeName: CHOLINE ION / type: ligand / ID: 2 / Number of copies: 3 / Formula: CHT
Molecular weightTheoretical: 104.171 Da
Chemical component information

ChemComp-CHT:
CHOLINE ION

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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

Concentration6 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
20.0 mMC4H11NO3Tris-base
100.0 mMC5H14ClNOcholine chloride
1.0 mMC9H15O6PTCEP
0.086 mMC56H92O25Digitonin glyco diosgenin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot 3s.
DetailsThis sample was mono disperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1918723
Startup modelType of model: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 435398
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationNumber classes: 8 / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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