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- EMDB-12566: Structure of human excitatory amino acid transporter 3 (EAAT3) in... -

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Entry
Database: EMDB / ID: EMD-12566
TitleStructure of human excitatory amino acid transporter 3 (EAAT3) in complex with HIP-B
Map data
Sample
  • Complex: Ternary complex of excitatory amino acid transporter 3 with inhibitor HIP-B
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: (+)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid
  • Ligand: (-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
KeywordsGLUTAMATE / ASPARTATE TRANSPORTER / CYSTEINE / INHIBITOR COMPLEX / MEMBRANE PROTEIN / Structural Genomics / Structural Genomics Consortium / SGC
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: 3.34 Å
AuthorsBaronina A / Pike ACW
Funding support United Kingdom, 2 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC) United Kingdom
Wellcome Trust106169/Z/14/Z United Kingdom
CitationJournal: TO BE PUBLISHED
Title: Structure of human excitatory amino acid transporter 3 (EAAT3) in complex with HIP-B
Authors: Baronina A / Pike ACW / Yu X / Dong YY / Shintre CA / Tessitore A / Chu A / Rotty B / Venkaya S / Mukhopadhyay SMM / Borkowska O / Chalk R / Shrestha L / Burgess-Brown NA / Edwards AM / ...Authors: Baronina A / Pike ACW / Yu X / Dong YY / Shintre CA / Tessitore A / Chu A / Rotty B / Venkaya S / Mukhopadhyay SMM / Borkowska O / Chalk R / Shrestha L / Burgess-Brown NA / Edwards AM / Arrowsmith CH / Bountra C / Han S / Carpenter EP
History
DepositionMar 5, 2021-
Header (metadata) releaseMar 16, 2022-
Map releaseMar 16, 2022-
UpdateJul 10, 2024-
Current statusJul 10, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7nsg
  • Surface level: 0.03
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_12566.png

Downloads & links

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Map

FileDownload / File: emd_12566.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.09 Å/pix.
x 200 pix.
= 217.2 Å		1.09 Å/pix.
x 200 pix.
= 217.2 Å		1.09 Å/pix.
x 200 pix.
= 217.2 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.086 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0252 / Movie #1: 0.03
Minimum - Maximum-0.20561619 - 0.2551124
Average (Standard dev.)0.00003249621 (±0.007815577)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 217.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0861.0861.086
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z217.200217.200217.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.2060.2550.000

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Supplemental data

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Mask #1

Fileemd_12566_msk_1.map
Projections & Slices
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Additional map: #1

Fileemd_12566_additional_1.map
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Half map: #1

Fileemd_12566_half_map_1.map
Projections & Slices
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Half map: #2

Fileemd_12566_half_map_2.map
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Sample components

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Entire : Ternary complex of excitatory amino acid transporter 3 with inhib...

EntireName: Ternary complex of excitatory amino acid transporter 3 with inhibitor HIP-B
Components
  • Complex: Ternary complex of excitatory amino acid transporter 3 with inhibitor HIP-B
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: (+)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid
  • Ligand: (-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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Supramolecule #1: Ternary complex of excitatory amino acid transporter 3 with inhib...

SupramoleculeName: Ternary complex of excitatory amino acid transporter 3 with inhibitor HIP-B
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 160 KDa

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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: 3 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 53.252867 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGKPARKGCE WKRFLKNNWV LLSTVAAVVL GITTGVLVRE HRNLSTLEKF YFAFPGEILM RMLKLIILPL IISSMITGVA ALDSNVSGK IGVRAVVYYF CTTLIAVILG IVLVVSIKPG VTQKVGEIAR TGSTPEVSTV DAMLDLIRNM FPENLVQACF Q QYKTKREE ...String:
MGKPARKGCE WKRFLKNNWV LLSTVAAVVL GITTGVLVRE HRNLSTLEKF YFAFPGEILM RMLKLIILPL IISSMITGVA ALDSNVSGK IGVRAVVYYF CTTLIAVILG IVLVVSIKPG VTQKVGEIAR TGSTPEVSTV DAMLDLIRNM FPENLVQACF Q QYKTKREE VKPPSDPEMN MTEESFTAVM TTAISKNKTK EYKIVGMYSD GINVLGLIVF CLVFGLVIGK MGEKGQILVD FF NALSDAT MKIVQIIMCY MPLGILFLIA GKIIEVEDWE IFRKLGLYMA TVLTGLAIHS IVILPLIYFI VVRKNPFRFA MGM AQALLT ALMISSSSAT LPVTFRCAEE NNQVDKRITR FVLPVGATIN MDGTALYEAV AAVFIAQLND LDLGIGQIIT ISIT ATSAS IGAAGVPQAG LVTMVIVLSA VGLPAEDVTL IIAVDWLLDR FRTMVNVLGD AFGTGIVEKL SKKELEQMDV SSEVN AENL YFQ

UniProtKB: Excitatory amino acid transporter 3

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Macromolecule #2: (+)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-c...

MacromoleculeName: (+)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid
type: ligand / ID: 2 / Number of copies: 3 / Formula: UR5
Molecular weightTheoretical: 172.139 Da
Chemical component information

ChemComp-UR5:
(+)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid

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Macromolecule #3: (-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-c...

MacromoleculeName: (-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid
type: ligand / ID: 3 / Number of copies: 3 / Formula: UR8
Molecular weightTheoretical: 172.139 Da
Chemical component information

ChemComp-UR8:
(-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid

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Macromolecule #4: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 3 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #5: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 5 / Number of copies: 15 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

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

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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

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
20.0 mMHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
0.003 %LMNGlauryl maltose neopentyl glycol
0.0003 %CHScholesteryl hemisuccinate

Details: Solutions were made fresh from pre-made, sterile filtered stocks.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: Blot time 4s Blot force -15 sample volume 3 uL.
DetailsMonodisperse sample after SEC

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 1 / Number real images: 4947 / Average electron dose: 42.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.6 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1169680
Startup modelType of model: NONE / Details: Ab-initio models were generated using RELION 2.1
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 127936
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final 3D classificationSoftware - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6s3q


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsMODEL REFINED USING NCS CONSTRAINTS AND ROTAMER, CBETA, SECONDARY STRUCTURE AND RAMACHANDARN RESTRAINTS AGAINST RELION POST-PROCESSED B-FACTOR SHARPENED (-118A**2) 3.34A FILTERED MAP
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-7nsg:
Structure of human excitatory amino acid transporter 3 (EAAT3) in complex with HIP-B

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