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- EMDB-26985: Human excitatory amino acid transporter 3 (EAAT3) with bound glut... -

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Basic information

Entry
Database: EMDB / ID: EMD-26985
TitleHuman excitatory amino acid transporter 3 (EAAT3) with bound glutamate in an intermediate outward facing state
Map dataExcitatory amino acid transporter 3 with glutamate bound at new intermediate outward facing states
Sample
  • Complex: Human EAAT3 with glutamate bound
    • Complex: Human EAAT3 with glutamate bound
      • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: GLUTAMIC ACID
  • Ligand: SODIUM IONSodium
  • Ligand: MERCURY (II) ION
  • Ligand: water
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 / grooming behavior / L-aspartate import across plasma membrane / 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 / blood vessel morphogenesis / motor neuron apoptotic process / motor behavior / glutamate binding / dopamine receptor signaling pathway / positive regulation of heart rate / adult behavior / maintenance of blood-brain barrier / 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 / axon terminus / monoatomic ion transport / synaptic cleft / chloride transmembrane transport / response to amphetamine / neurogenesis / locomotory behavior / dendritic shaft / 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 / cellular response to oxidative stress / late endosome membrane / gene expression / 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 / neuronal cell body / dendrite / 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: 2.8 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 13, 2022-
Header (metadata) releaseMay 10, 2023-
Map releaseMay 10, 2023-
UpdateMay 17, 2023-
Current statusMay 17, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26985.png

Downloads & links

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Map

FileDownload / File: emd_26985.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationExcitatory amino acid transporter 3 with glutamate bound at new intermediate outward facing states
Voxel sizeX=Y=Z: 1.083 Å
Density
Contour LevelBy AUTHOR: 0.8
Minimum - Maximum-4.7072587 - 7.69233
Average (Standard dev.)0.0118373055 (±0.14662811)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 277.248 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_26985_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_26985_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 glutamate bound

EntireName: Human EAAT3 with glutamate bound
Components
  • Complex: Human EAAT3 with glutamate bound
    • Complex: Human EAAT3 with glutamate bound
      • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: GLUTAMIC ACID
  • Ligand: SODIUM IONSodium
  • Ligand: MERCURY (II) ION
  • Ligand: water

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Supramolecule #1: Human EAAT3 with glutamate bound

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

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Supramolecule #2: Human EAAT3 with glutamate bound

SupramoleculeName: Human EAAT3 with glutamate bound / type: complex / ID: 2 / Chimera: Yes / 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

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Macromolecule #2: GLUTAMIC ACID

MacromoleculeName: GLUTAMIC ACID / type: ligand / ID: 2 / Number of copies: 3 / Formula: GLU
Molecular weightTheoretical: 147.129 Da
Chemical component information

ChemComp-GLU:
GLUTAMIC ACID / Glutamic acid

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 9
Molecular weightTheoretical: 22.99 Da

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Macromolecule #4: MERCURY (II) ION

MacromoleculeName: MERCURY (II) ION / type: ligand / ID: 4 / Number of copies: 3 / Formula: HG
Molecular weightTheoretical: 200.59 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 12 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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

Concentration4.6 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
20.0 mMC8H18N2O4SHepes
200.0 mMNaClSodium chloridesodium chloride
0.086 mMC56H92O25Digitonin glyco diosgenin
20.0 mMC5H9NO4L-glutamateGlutamic acid

Details: Tris is used for 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.
DetailsHuman EAAT3 with glutamate bound

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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 / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 81000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.266 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 8511485
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 8 / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 496972
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:

6x2z

,

6x2l

)
RefinementProtocol: RIGID BODY FIT
Output model

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

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