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- EMDB-26997: Human excitatory amino acid transporter 3 (EAAT3) with bound pota... -

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Basic information

Entry
Database: EMDB / ID: EMD-26997
TitleHuman excitatory amino acid transporter 3 (EAAT3) with bound potassium in an intermediate outward facing state
Map datahuman Excitatory amino acid transporter 3 with potassium bound at intermediate outward facing (iOFS)state
Sample
  • Complex: Human EAAT3 with potassium bound
    • Complex: Human EAAT3 with potassium bound
      • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: POTASSIUM ION
  • Ligand: MERCURY (II) ION
  • Ligand: water
KeywordsTRANSPORT 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 / cysteine transport / cysteine transmembrane transporter activity / neurotransmitter receptor transport to plasma membrane / 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 / cysteine transport / cysteine transmembrane transporter activity / neurotransmitter receptor transport to plasma membrane / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / response to decreased oxygen levels / L-glutamate import / cellular response to mercury ion / Transport of inorganic cations/anions and amino acids/oligopeptides / retina layer formation / L-glutamate transmembrane transporter activity / cellular response to ammonium ion / L-glutamate transmembrane transport / glutathione biosynthetic process / D-aspartate import across plasma membrane / L-aspartate transmembrane transport / righting reflex / zinc ion transmembrane transport / cellular response to bisphenol A / L-aspartate transmembrane transporter activity / grooming behavior / intracellular glutamate homeostasis / L-aspartate import across plasma membrane / Glutamate Neurotransmitter Release Cycle / proximal dendrite / monoatomic anion channel activity / L-glutamate import across plasma membrane / apical dendrite / transepithelial transport / response to anesthetic / conditioned place preference / intracellular zinc ion homeostasis / cellular response to cocaine / chloride transmembrane transporter activity / blood vessel morphogenesis / motor neuron apoptotic process / response to morphine / G protein-coupled dopamine receptor signaling pathway / motor behavior / glutamate receptor signaling pathway / glutamate binding / neurotransmitter transport / superoxide metabolic process / heart contraction / maintenance of blood-brain barrier / perisynaptic space / dopamine metabolic process / adult behavior / : / asymmetric synapse / glial cell projection / response to axon injury / behavioral fear response / synaptic cleft / postsynaptic modulation of chemical synaptic transmission / transport across blood-brain barrier / positive regulation of heart rate / monoatomic ion transport / neurogenesis / axon terminus / response to amphetamine / chloride transmembrane transport / dendritic shaft / cell periphery / locomotory behavior / synapse organization / cytokine-mediated signaling pathway / brain development / Schaffer collateral - CA1 synapse / memory / long-term synaptic potentiation / recycling endosome membrane / late endosome membrane / presynapse / cellular response to oxidative stress / early endosome membrane / gene expression / chemical synaptic transmission / dendritic spine / perikaryon / negative regulation of neuron apoptotic process / apical plasma membrane / membrane raft / response to xenobiotic stimulus / axon / external side of plasma membrane / neuronal cell body / dendrite / endoplasmic reticulum / extracellular exosome / metal ion binding / identical protein binding / membrane / 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: 2.44 Å
AuthorsQiu B / Boudker O
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS085318 United States
CitationJournal: Nat Commun / Year: 2023
Title: Symport and antiport mechanisms of human glutamate transporters.
Authors: Biao Qiu / Olga Boudker /
Abstract: Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and ...Excitatory amino acid transporters (EAATs) uptake glutamate into glial cells and neurons. EAATs achieve million-fold transmitter gradients by symporting it with three sodium ions and a proton, and countertransporting a potassium ion via an elevator mechanism. Despite the availability of structures, the symport and antiport mechanisms still need to be clarified. We report high-resolution cryo-EM structures of human EAAT3 bound to the neurotransmitter glutamate with symported ions, potassium ions, sodium ions alone, or without ligands. We show that an evolutionarily conserved occluded translocation intermediate has a dramatically higher affinity for the neurotransmitter and the countertransported potassium ion than outward- or inward-facing transporters and plays a crucial role in ion coupling. We propose a comprehensive ion coupling mechanism involving a choreographed interplay between bound solutes, conformations of conserved amino acid motifs, and movements of the gating hairpin and the substrate-binding domain.
History
DepositionMay 17, 2022-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateOct 9, 2024-
Current statusOct 9, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26997.png

Downloads & links

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Map

FileDownload / File: emd_26997.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman Excitatory amino acid transporter 3 with potassium bound at intermediate outward facing (iOFS)state
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 300 pix.
= 255.6 Å		0.85 Å/pix.
x 300 pix.
= 255.6 Å		0.85 Å/pix.
x 300 pix.
= 255.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.852 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.55
Minimum - Maximum-3.336921 - 4.712938
Average (Standard dev.)0.0077160015 (±0.10373889)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 255.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_26997_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_26997_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human EAAT3 with potassium bound

EntireName: Human EAAT3 with potassium bound
Components
  • Complex: Human EAAT3 with potassium bound
    • Complex: Human EAAT3 with potassium bound
      • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: POTASSIUM ION
  • Ligand: MERCURY (II) ION
  • Ligand: water

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Supramolecule #1: Human EAAT3 with potassium bound

SupramoleculeName: Human EAAT3 with potassium bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: Human EAAT3 with potassium bound

SupramoleculeName: Human EAAT3 with potassium bound / type: complex / ID: 2 / Parent: 1 / 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 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 57.120863 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPMGKPARKG AEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PLIISSMITG VAALDSNVS GKIGLRAVVY YFATTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVDAMLDLIR NMFPENLVQA A FQQYKTKR ...String:
GPMGKPARKG AEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PLIISSMITG VAALDSNVS GKIGLRAVVY YFATTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVDAMLDLIR NMFPENLVQA A FQQYKTKR EEVKPPSDPE MTMTEESFTA VMTTAISKTK TKEYKIVGMY SDGINVLGLI VFALVFGLVI GKMGEKGQIL VD FFNALSD ATMKIVQIIM WYMPLGILFL IAGCIIEVED WEIFRKLGLY MATVLTGLAI HSIVILPLIY FIVVRKNPFR FAM GMAQAL LTALMISSSS ATLPVTFRCA EENNQVDKRI TRFVLPVGAT INMDGTALYE AVAAVFIAQL NDLDLGIGQI ITIS ITATS ASIGAAGVPQ AGLVTMVIVL SAVGLPAEDV TLIIAVDCLL DRFRTMVNVL GDAFGTGIVE KLSKKELEQM DVSSE VNIV NPFALESTIL DNEDSDTKKS YVNGGFAVDK SDTISFTQTS QF

UniProtKB: Excitatory amino acid transporter 3

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Macromolecule #2: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 2 / Number of copies: 3 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #3: MERCURY (II) ION

MacromoleculeName: MERCURY (II) ION / type: ligand / ID: 3 / Number of copies: 3 / Formula: HG
Molecular weightTheoretical: 200.59 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 30 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration6.2 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHepes
300.0 mMKClpotassium chloride
0.086 mMC56H92O25Digitonin glyco diosgenin

Details: Tris is used fot adjusting pH
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.
Detailsthe sample was monodisperse

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 51.1 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 / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.3 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: 3502663
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.44 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 404729
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 8 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChain

6x2z

source_name: PDB, initial_model_type: experimental model

6x3e

source_name: PDB, initial_model_type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8cua:
Human excitatory amino acid transporter 3 (EAAT3) with bound potassium in an intermediate outward facing state

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