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- EMDB-27007: Human Excitatory excitatory amino acid transporter 3 (EAAT3) prot... -

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Basic information

Entry
Database: EMDB / ID: EMD-27007
TitleHuman Excitatory excitatory amino acid transporter 3 (EAAT3) protomer at in an intermediate outward facing sodium-bound state
Map datahuman Excitatory amino acid transporter3 protomer at intermediate outward facing (iOFS) sodium bound state
Sample
  • Complex: human Excitatory amino acid transporter 3 at outward facing (OFS) sodium bound state
    • Complex: human Excitatory amino acid transporter 3 at outward facing (OFS) sodium bound state
      • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: SODIUM ION
KeywordsTRANSPORT PROTEIN
Function / homology
Function and homology information


D-aspartate transmembrane transport / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / L-cysteine transport / L-cysteine transmembrane transporter activity / L-glutamate import / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / SLC-mediated transport of amino acids ...D-aspartate transmembrane transport / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / distal dendrite / L-cysteine transport / L-cysteine transmembrane transporter activity / L-glutamate import / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / SLC-mediated transport of amino acids / L-glutamate transmembrane transporter activity / glutathione biosynthetic process / grooming behavior / L-glutamate transmembrane transport / L-aspartate transmembrane transport / zinc ion transmembrane transport / L-aspartate transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-aspartate import across plasma membrane / D-aspartate import across plasma membrane / blood vessel morphogenesis / monoatomic anion channel activity / proximal dendrite / L-glutamate import across plasma membrane / intracellular zinc ion homeostasis / motor behavior / transepithelial transport / cellular response to cocaine / apical dendrite / chloride transmembrane transporter activity / G protein-coupled dopamine receptor signaling pathway / superoxide metabolic process / adult behavior / response to morphine / maintenance of blood-brain barrier / perisynaptic space / dopamine metabolic process / retina development in camera-type eye / glutamate receptor signaling pathway / neurotransmitter transport / amino acid transport / asymmetric synapse / glial cell projection / postsynaptic modulation of chemical synaptic transmission / conditioned place preference / positive regulation of heart rate / neurogenesis / synaptic cleft / transport across blood-brain barrier / response to amphetamine / axon terminus / chloride transmembrane transport / locomotory behavior / cell periphery / dendritic shaft / brain development / memory / synapse organization / recycling endosome / neuron apoptotic process / Schaffer collateral - CA1 synapse / gene expression / recycling endosome membrane / cytokine-mediated signaling pathway / apical part of cell / late endosome membrane / presynapse / chemical synaptic transmission / early endosome membrane / negative regulation of neuron apoptotic process / dendritic spine / perikaryon / apical plasma membrane / membrane raft / axon / neuronal cell body / synapse / dendrite / cell surface / extracellular exosome / membrane / metal ion binding / identical protein 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: 3.04 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-
UpdateJun 12, 2024-
Current statusJun 12, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27007.png

Downloads & links

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Map

FileDownload / File: emd_27007.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationhuman Excitatory amino acid transporter3 protomer at intermediate outward facing (iOFS) sodium bound 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: 0.022
Minimum - Maximum-0.099496864 - 0.19109586
Average (Standard dev.)-0.0000030215322 (±0.0043469756)
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_27007_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_27007_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 Excitatory amino acid transporter 3 at outward facing (OFS)...

EntireName: human Excitatory amino acid transporter 3 at outward facing (OFS) sodium bound state
Components
  • Complex: human Excitatory amino acid transporter 3 at outward facing (OFS) sodium bound state
    • Complex: human Excitatory amino acid transporter 3 at outward facing (OFS) sodium bound state
      • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: SODIUM ION

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Supramolecule #1: human Excitatory amino acid transporter 3 at outward facing (OFS)...

SupramoleculeName: human Excitatory amino acid transporter 3 at outward facing (OFS) sodium bound state
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #2: human Excitatory amino acid transporter 3 at outward facing (OFS)...

SupramoleculeName: human Excitatory amino acid transporter 3 at outward facing (OFS) sodium bound state
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: 1 / 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: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 2 / Number of copies: 2
Molecular weightTheoretical: 22.99 Da

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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

Concentration4.1 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHepes
300.0 mMNaClSodium Chloride
0.086 mMC56H92O25Digitonin glyco diosgenin

Details: Tris is used to adjust pH. Not list here because the concentration is unknown
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: 57.52 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: 2949270
Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.04 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Details: This is the number of C3 expanded protomer / Number images used: 105451
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final 3D classificationNumber classes: 10 / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6x2z


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-8cv3:
Human Excitatory excitatory amino acid transporter 3 (EAAT3) protomer at in an intermediate outward facing sodium-bound state

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