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- PDB-7vr7: Inward-facing structure of human EAAT2 in the WAY213613-bound state -

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Basic information

Entry
Database: PDB / ID: 7vr7
TitleInward-facing structure of human EAAT2 in the WAY213613-bound state
ComponentsExcitatory amino acid transporter 2
KeywordsTRANSPORT PROTEIN / transporter / MEMBRANE PROTEIN
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 / : / telencephalon development / glutathione biosynthetic process / L-aspartate import across plasma membrane / neutral L-amino acid transmembrane transporter activity / monoatomic anion transmembrane transporter activity / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / transepithelial transport / astrocyte projection / neuron projection terminus / neurotransmitter 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
CHOLESTEROL / Chem-GJ0 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Excitatory amino acid transporter 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.49 Å
AuthorsKato, T. / Kusakizako, T. / Yamashita, K. / Nishizawa, T. / Nureki, O.
Funding support Japan, 1items
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, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 10, 2022Provider: repository / Type: Initial release
Revision 1.1Sep 28, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Jun 26, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_admin / refine
Item: _em_admin.last_update / _refine.ls_d_res_high / _refine.ls_d_res_low

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Excitatory amino acid transporter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)66,3307
Polymers63,0171
Non-polymers3,3146
Water00
1
A: Excitatory amino acid transporter 2
hetero molecules

A: Excitatory amino acid transporter 2
hetero molecules

A: Excitatory amino acid transporter 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,99121
Polymers189,0513
Non-polymers9,94118
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation2
Noncrystallographic symmetry (NCS)NCS oper:
IDCodeMatrixVector
1given(1), (1), (1)
2generate(-0.5, -0.866025404), (0.866025404, -0.5), (1)353.48414, 94.71584
3generate(-0.5, 0.866025404), (-0.866025404, -0.5), (1)94.71584, 353.48414

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Components

#1: Protein Excitatory amino acid transporter 2 / Glutamate/aspartate transporter II / Sodium-dependent glutamate/aspartate transporter 2 / Solute ...Glutamate/aspartate transporter II / Sodium-dependent glutamate/aspartate transporter 2 / Solute carrier family 1 member 2


Mass: 63016.879 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A2, EAAT2, GLT1 / Production host: Homo sapiens (human) / References: UniProt: P43004
#2: Chemical ChemComp-GJ0 / (2S)-2-azanyl-4-[[4-[2-bromanyl-4,5-bis(fluoranyl)phenoxy]phenyl]amino]-4-oxidanylidene-butanoic acid / WAY-213613


Mass: 415.186 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C16H13BrF2N2O4 / Feature type: SUBJECT OF INVESTIGATION / Comment: inhibitor*YM
#3: Chemical ChemComp-9Z9 / (3beta,14beta,17beta,25R)-3-[4-methoxy-3-(methoxymethyl)butoxy]spirost-5-en


Mass: 544.805 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C34H56O5 / Comment: detergent*YM
#4: Chemical ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE


Mass: 790.145 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: EAAT2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER/RHODIUM / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 1600 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: REFMAC / Version: 5.8.0291 / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
7MOLREPmodel fitting
9REFMAC5.8.0291model refinement
13RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.49 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 527996 / Symmetry type: POINT
RefinementResolution: 3.49→3.49 Å / Cor.coef. Fo:Fc: 0.848 / WRfactor Rwork: 0.345 / SU B: 9.696 / SU ML: 0.184 / Average fsc overall: 0.8451 / Average fsc work: 0.8451 / ESU R: 0.176
Details: Hydrogens have been added in their riding positions
RfactorNum. reflection% reflection
Rwork0.3452 108617 -
obs--100 %
Solvent computationSolvent model: BABINET MODEL
Displacement parametersBiso mean: 85.71 Å2
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0110.0133245
ELECTRON MICROSCOPYr_bond_other_d0.0070.0173397
ELECTRON MICROSCOPYr_ext_dist_refined_b0.0580.1319
ELECTRON MICROSCOPYr_angle_refined_deg1.6891.6224413
ELECTRON MICROSCOPYr_angle_other_deg1.5931.597798
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.7565398
ELECTRON MICROSCOPYr_dihedral_angle_2_deg25.96221.869107
ELECTRON MICROSCOPYr_dihedral_angle_3_deg18.18615534
ELECTRON MICROSCOPYr_dihedral_angle_4_deg9.4161512
ELECTRON MICROSCOPYr_chiral_restr0.10.2461
ELECTRON MICROSCOPYr_gen_planes_refined0.0130.023407
ELECTRON MICROSCOPYr_gen_planes_other0.0080.02655
ELECTRON MICROSCOPYr_nbd_refined0.2160.21820
ELECTRON MICROSCOPYr_symmetry_nbd_other0.1510.27158
ELECTRON MICROSCOPYr_nbtor_refined0.170.23306
ELECTRON MICROSCOPYr_symmetry_nbtor_other0.0730.23642
ELECTRON MICROSCOPYr_xyhbond_nbd_refined0.130.2226
ELECTRON MICROSCOPYr_symmetry_nbd_refined0.1510.218
ELECTRON MICROSCOPYr_nbd_other0.1640.2158
ELECTRON MICROSCOPYr_symmetry_xyhbond_nbd_refined0.0220.22
ELECTRON MICROSCOPYr_mcbond_it3.6438.4421604
ELECTRON MICROSCOPYr_mcbond_other3.6418.4351603
ELECTRON MICROSCOPYr_mcangle_it6.37812.6321998
ELECTRON MICROSCOPYr_mcangle_other6.37712.6411999
ELECTRON MICROSCOPYr_scbond_it3.99.8281641
ELECTRON MICROSCOPYr_scbond_other3.8999.8321642
ELECTRON MICROSCOPYr_scangle_it6.92914.4342415
ELECTRON MICROSCOPYr_scangle_other6.92814.4382416
ELECTRON MICROSCOPYr_lrange_it19.659164.62614301
ELECTRON MICROSCOPYr_lrange_other19.659164.62814302
LS refinement shell

Refine-ID: ELECTRON MICROSCOPY / Num. reflection Rfree: _ / Total num. of bins used: 20 / % reflection obs: 100 %

Resolution (Å)Rfactor RworkNum. reflection RworkRfactor allNum. reflection allFsc workWRfactor Rwork
2.8-2.8731.42781261.42781260.571.427
2.873-2.9510.7877450.7877450.6660.78
2.951-3.0370.61476760.61476760.7360.614
3.037-3.130.4873890.4873890.8010.48
3.13-3.2330.39370850.39370850.8320.393
3.233-3.3460.31969650.31969650.8710.319
3.346-3.4730.28267450.28267450.8960.282
3.473-3.6140.26464080.26464080.9110.264
3.614-3.7750.26361430.26361430.9280.263
3.775-3.9590.2658570.2658570.9420.26
3.959-4.1730.26956390.26956390.9440.269
4.173-4.4250.28553730.28553730.9510.285
4.425-4.7310.28149140.28149140.9510.281
4.731-5.1090.26546430.26546430.9480.265
5.109-5.5960.26343230.26343230.9190.263
5.596-6.2540.35938380.35938380.8690.359
6.254-7.2180.37534230.37534230.830.375
7.218-8.8330.32128680.32128680.870.321
8.833-12.4570.26622210.26622210.9190.266
12.457-119.520.7112360.7112360.9660.71

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