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- EMDB-32098: Inward-facing structure of human EAAT2 in the substrate-free state -

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Basic information

Entry
Database: EMDB / ID: EMD-32098
TitleInward-facing structure of human EAAT2 in the substrate-free state
Map data
Sample
  • Complex: EAAT2
    • Protein or peptide: Excitatory amino acid transporter 2
  • Ligand: (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
Function / homology
Function and homology information


neurotransmitter reuptake / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / cysteine transmembrane transporter activity / high-affinity L-glutamate transmembrane transporter activity / visual behavior / glutamate:sodium symporter activity / Transport of inorganic cations/anions and amino acids/oligopeptides / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity ...neurotransmitter reuptake / Astrocytic Glutamate-Glutamine Uptake And Metabolism / membrane protein complex / cysteine transmembrane transporter activity / high-affinity L-glutamate transmembrane transporter activity / visual behavior / glutamate:sodium symporter activity / Transport of inorganic cations/anions and amino acids/oligopeptides / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / L-aspartate transmembrane transport / D-aspartate import across plasma membrane / glutathione biosynthetic process / neutral L-amino acid transmembrane transporter activity / : / telencephalon development / L-aspartate import across plasma membrane / monoatomic anion transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / astrocyte projection / neuron projection terminus / neurotransmitter transport / transepithelial transport / cellular response to cocaine / adult behavior / protein homotrimerization / transport across blood-brain barrier / axolemma / response to amino acid / monoatomic ion transport / positive regulation of glucose import / multicellular organism growth / response to wounding / presynaptic membrane / cell body / chemical synaptic transmission / vesicle / response to xenobiotic stimulus / membrane raft / glutamatergic synapse / cell surface / membrane / 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 2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.58 Å
AuthorsKato T / Kusakizako T / Yamashita K / Nishizawa T / Nureki O
Funding support Japan, 1 items
OrganizationGrant numberCountry
Japan Society for the Promotion of Science (JSPS) Japan
CitationJournal: Nat Commun / Year: 2022
Title: Structural insights into inhibitory mechanism of human excitatory amino acid transporter EAAT2.
Authors: Takafumi Kato / Tsukasa Kusakizako / Chunhuan Jin / Xinyu Zhou / Ryuichi Ohgaki / LiLi Quan / Minhui Xu / Suguru Okuda / Kan Kobayashi / Keitaro Yamashita / Tomohiro Nishizawa / Yoshikatsu ...Authors: Takafumi Kato / Tsukasa Kusakizako / Chunhuan Jin / Xinyu Zhou / Ryuichi Ohgaki / LiLi Quan / Minhui Xu / Suguru Okuda / Kan Kobayashi / Keitaro Yamashita / Tomohiro Nishizawa / Yoshikatsu Kanai / Osamu Nureki /
Abstract: Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial ...Glutamate is a pivotal excitatory neurotransmitter in mammalian brains, but excessive glutamate causes numerous neural disorders. Almost all extracellular glutamate is retrieved by the glial transporter, Excitatory Amino Acid Transporter 2 (EAAT2), belonging to the SLC1A family. However, in some cancers, EAAT2 expression is enhanced and causes resistance to therapies by metabolic disturbance. Despite its crucial roles, the detailed structural information about EAAT2 has not been available. Here, we report cryo-EM structures of human EAAT2 in substrate-free and selective inhibitor WAY213613-bound states at 3.2 Å and 2.8 Å, respectively. EAAT2 forms a trimer, with each protomer consisting of transport and scaffold domains. Along with a glutamate-binding site, the transport domain possesses a cavity that could be disrupted during the transport cycle. WAY213613 occupies both the glutamate-binding site and cavity of EAAT2 to interfere with its alternating access, where the sensitivity is defined by the inner environment of the cavity. We provide the characterization of the molecular features of EAAT2 and its selective inhibition mechanism that may facilitate structure-based drug design for EAAT2.
History
DepositionOct 22, 2021-
Header (metadata) releaseAug 10, 2022-
Map releaseAug 10, 2022-
UpdateSep 28, 2022-
Current statusSep 28, 2022Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_32098.png

Downloads & links

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Map

FileDownload / File: emd_32098.map.gz / Format: CCP4 / Size: 7.3 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.245 Å
Density
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.2272078 - 0.3464862
Average (Standard dev.)0.0014958612 (±0.0107268635)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin585858
Dimensions124124124
Spacing124124124
CellA=B=C: 154.38 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_32098_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_32098_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_32098_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : EAAT2

EntireName: EAAT2
Components
  • Complex: EAAT2
    • Protein or peptide: Excitatory amino acid transporter 2
  • Ligand: (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en
  • Ligand: CHOLESTEROL
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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Supramolecule #1: EAAT2

SupramoleculeName: EAAT2 / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)

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Macromolecule #1: Excitatory amino acid transporter 2

MacromoleculeName: Excitatory amino acid transporter 2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 63.016879 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MASTEGANNM PKQVEVRMHD SHLGSEEPKH RHLGLRLCDK LGKNLLLTLT VFGVILGAVC GGLLRLASPI HPDVVMLIAF PGDILMRML KMLILPLIIS SLITGLSGLD AKASGRLGTR AMVYYMSTTI IAAVLGVILV LAIHPGNPKL KKQLGPGKKN D EVSSLDAF ...String:
MASTEGANNM PKQVEVRMHD SHLGSEEPKH RHLGLRLCDK LGKNLLLTLT VFGVILGAVC GGLLRLASPI HPDVVMLIAF PGDILMRML KMLILPLIIS SLITGLSGLD AKASGRLGTR AMVYYMSTTI IAAVLGVILV LAIHPGNPKL KKQLGPGKKN D EVSSLDAF LDLIRNLFPE NLVQACFQQI QTVTKKVLVA PPPDEEANAT SAVVSLLNET VTEVPEETKM VIKKGLEFKD GM NVLGLIG FFIAFGIAMG KMGDQAKLMV DFFNILNEIV MKLVIMIMWY SPLGIACLIC GKIIAIKDLE VVARQLGMYM VTV IIGLII HGGIFLPLIY FVVTRKNPFS FFAGIFQAWI TALGTASSAG TLPVTFRCLE ENLGIDKRVT RFVLPVGATI NMDG TALYE AVAAIFIAQM NGVVLDGGQI VTVSLTATLA SVGAASIPSA GLVTMLLILT AVGLPTEDIS LLVAVDWLLD RMRTS VNVV GDSFGAGIVY HLSKSELDTI DSQHRVHEDI EMTKTQSIYD DMKNHRESNS NQCVYAAHNS VIVDECKVTL AANGKS ADC SVEEEPWKRE KENLYFQG

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Macromolecule #2: (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]sp...

MacromoleculeName: (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en
type: ligand / ID: 2 / Number of copies: 1 / Formula: 9Z9
Molecular weightTheoretical: 544.805 Da
Chemical component information

ChemComp-9Z9:
(3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en / detergent*YM

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Macromolecule #3: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 3 / Number of copies: 1 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL / Cholesterol

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Macromolecule #4: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 4 / Number of copies: 2 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM / Phosphatidylcholine

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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
GridModel: Quantifoil R1.2/1.3 / Material: COPPER/RHODIUM / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1.6 µm / Nominal defocus min: 0.8 µm
Specialist opticsEnergy filter - Name: GIF Bioquantum
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.58 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 212554
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6gct

RefinementSpace: RECIPROCAL
Output model

PDB-7vr8:
Inward-facing structure of human EAAT2 in the substrate-free state

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