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- EMDB-46592: Human excitatory amino acid transporter 3 (EAAT3) monomer in 200 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-46592
TitleHuman excitatory amino acid transporter 3 (EAAT3) monomer in 200 mM NaCl, 10 mM L-Cysteine at IFS
Map dataEAAT3 monomer at IFS in 200 mM NaCl, 10 mM L-Cysteine
Sample
  • Complex: human excitatory amino acid transporter 3
    • Protein or peptide: human Human excitatory amino acid transporter 3
Keywordshuman EAAT3 / IFS / sodium bound / TRANSPORT PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 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

Downloads & links

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Map

FileDownload / File: emd_46592.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEAAT3 monomer at IFS in 200 mM NaCl, 10 mM L-Cysteine
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
Contour LevelBy AUTHOR: 0.43
Minimum - Maximum-4.464724 - 6.3331957
Average (Standard dev.)0.005562926 (±0.112203695)
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_46592_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map1

Fileemd_46592_half_map_1.map
Annotationhalf map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: half map2

Fileemd_46592_half_map_2.map
Annotationhalf map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : human excitatory amino acid transporter 3

EntireName: human excitatory amino acid transporter 3
Components
  • Complex: human excitatory amino acid transporter 3
    • Protein or peptide: human Human excitatory amino acid transporter 3

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Supramolecule #1: human excitatory amino acid transporter 3

SupramoleculeName: human excitatory amino acid transporter 3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: human Human excitatory amino acid transporter 3

MacromoleculeName: human Human excitatory amino acid transporter 3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
SequenceString: GPMGKPARKG AEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PL IISSMIT GVAALDSNVS GKIGLRAVVY YFATTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVD AMLDLI RNMFPENLVQ AAFQQYKTKR ...String:
GPMGKPARKG AEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PL IISSMIT GVAALDSNVS GKIGLRAVVY YFATTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVD AMLDLI RNMFPENLVQ AAFQQYKTKR EEVKPPSDPE MTMTEESFTA VMTTAISKTK TKEYKIVGMY SDGI NVLGL IVFALVFGLV IGKMGEKGQI LVDFFNALSD ATMKIVQIIM WYMPLGILFL IAGCIIEVED WEIFR KLGL YMATVLTGLA IHSIVILPLI YFIVVRKNPF RFAMGMAQAL LTALMISSSS ATLPVTFRCA EENNQV DKR ITRFVLPVGA TINMDGTALY EAVAAVFIAQ LNDLDLGIGQ IITISITATS ASIGAAGVPQ AGLVTMV IV LSAVGLPAED VTLIIAVDCL LDRFRTMVNV LGDAFGTGIV EKLSKKELEQ MDVSSEVNIV NPFALEST I LDNEDSDTKK SYVNGGFAVD KSDTISFTQT SQF

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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

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: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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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: 2.94 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: 159538
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 local classification for monomers on expanded particles.
FSC plot (resolution estimation)

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