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- EMDB-46592: Human excitatory amino acid transporter 3 (EAAT3) monomer in 200 ... -
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Open data
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Basic information
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Title | Human excitatory amino acid transporter 3 (EAAT3) monomer in 200 mM NaCl, 10 mM L-Cysteine at IFS | |||||||||
![]() | EAAT3 monomer at IFS in 200 mM NaCl, 10 mM L-Cysteine | |||||||||
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![]() | human EAAT3 / IFS / sodium bound / TRANSPORT PROTEIN | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.94 Å | |||||||||
![]() | Qiu B / Boudker O | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 97.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 15.6 KB 15.6 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.9 KB | Display | ![]() |
Images | ![]() | 70.9 KB | ||
Masks | ![]() | 103 MB | ![]() | |
Filedesc metadata | ![]() | 5.5 KB | ||
Others | ![]() ![]() | 95.7 MB 95.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 1 MB | Display | ![]() |
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Full document | ![]() | 1 MB | Display | |
Data in XML | ![]() | 18.2 KB | Display | |
Data in CIF | ![]() | 23.5 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | EAAT3 monomer at IFS in 200 mM NaCl, 10 mM L-Cysteine | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.825 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
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Density Histograms |
-Half map: half map1
File | emd_46592_half_map_1.map | ||||||||||||
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Annotation | half map1 | ||||||||||||
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Density Histograms |
-Half map: half map2
File | emd_46592_half_map_2.map | ||||||||||||
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Annotation | half map2 | ||||||||||||
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Density Histograms |
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Sample components
-Entire : human excitatory amino acid transporter 3
Entire | Name: human excitatory amino acid transporter 3 |
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Components |
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-Supramolecule #1: human excitatory amino acid transporter 3
Supramolecule | Name: human excitatory amino acid transporter 3 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: human Human excitatory amino acid transporter 3
Macromolecule | Name: human Human excitatory amino acid transporter 3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Sequence | String: 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 |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.4 Details: 20 mM Hepes-Tris pH 7.4, 200 mM NaCl and 0.01% GDN, 10 mM L-Cysteine |
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K |
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Electron microscopy
Microscope | FEI/PHILIPS CM300FEG/T |
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Specialist optics | Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 58.25 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm |