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- EMDB-46590: Human excitatory amino acid transporter 3 (EAAT3) with bound L-Cy... -

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Basic information

Entry
Database: EMDB / ID: EMD-46590
TitleHuman excitatory amino acid transporter 3 (EAAT3) with bound L-Cysteine in an intermediate outward facing state (slight upper position)
Map dataEAAT3 monomer with L-Cys bound at iOFS
Sample
  • Complex: EAAT3 monomer with L-Cys bound at iOFS
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: CYSTEINE
  • Ligand: SODIUM ION
  • Ligand: MERCURY (II) ION
Keywordshuman EAAT3 / Cysteine / iOFS-upper / 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 / 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: 2.99 Å
AuthorsQiu B / Boudker O
Funding support United States, 2 items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS085318 United States
CitationJournal: bioRxiv / Year: 2024
Title: Structural basis of the excitatory amino acid transporter 3 substrate recognition.
Authors: Biao Qiu / Olga Boudker /
Abstract: Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only ...Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent work suggests that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate promiscuity by determining the cryo-EM structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures illustrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.
History
DepositionAug 14, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46590.png

Downloads & links

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Map

FileDownload / File: emd_46590.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEAAT3 monomer with L-Cys bound at iOFS
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 300 pix.
= 247.5 Å		0.83 Å/pix.
x 300 pix.
= 247.5 Å		0.83 Å/pix.
x 300 pix.
= 247.5 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.825 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.54
Minimum - Maximum-5.3313885 - 6.5441313
Average (Standard dev.)0.0056474465 (±0.14245723)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 247.5 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_46590_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map1

Fileemd_46590_half_map_1.map
Annotationhalf map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map2

Fileemd_46590_half_map_2.map
Annotationhalf map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EAAT3 monomer with L-Cys bound at iOFS

EntireName: EAAT3 monomer with L-Cys bound at iOFS
Components
  • Complex: EAAT3 monomer with L-Cys bound at iOFS
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: CYSTEINE
  • Ligand: SODIUM ION
  • Ligand: MERCURY (II) ION

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Supramolecule #1: EAAT3 monomer with L-Cys bound at iOFS

SupramoleculeName: EAAT3 monomer with L-Cys bound at iOFS / 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 / 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: CYSTEINE

MacromoleculeName: CYSTEINE / type: ligand / ID: 2 / Number of copies: 1 / Formula: CYS
Molecular weightTheoretical: 121.158 Da
Chemical component information

ChemComp-BTC:
CYSTEINE

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 3
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: 1 / Formula: HG
Molecular weightTheoretical: 200.59 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

Concentration5 mg/mL
BufferpH: 7.4
Details: 20 mM Hepes-Tris pH 7.4, 200 mM NaCl and 0.01% GDN, 10 mM L-Cysteine
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
DetailsThis sample was monodisperse.

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Electron microscopy

MicroscopeFEI/PHILIPS CM300FEG/T
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 58.25 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm

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Image processing

Particle selectionNumber selected: 4180263
Details: This is the number of trimer particle from the image
Startup modelType of model: NONE / Details: ab-initio
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3)
Details: This is number of monomer from C3 expanded particles.
Number images used: 60670
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3)
Final 3D classificationNumber classes: 20 / Software - Name: RELION (ver. 3)
Details: This is a local classification for monomers on expanded particles.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

8cv3


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: OTHER / Overall B value: 59.17
Output model

PDB-9d69:
Human excitatory amino acid transporter 3 (EAAT3) with bound L-Cysteine in an intermediate outward facing state (slight upper position)

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