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- EMDB-10094: Structure of human excitatory amino acid transporter 3 (EAAT3) in... -

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Basic information

Entry
Database: EMDB / ID: EMD-10094
TitleStructure of human excitatory amino acid transporter 3 (EAAT3) in complex with TFB-TBOA
Map dataPost-processed RELION map used for building and refinement
Sample
  • Organelle or cellular component: EXCITATORY AMINO ACID TRANSPORTER 3 TRIMER
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: (2~{S},3~{S})-2-azanyl-3-[[3-[[4-(trifluoromethyl)phenyl]carbonylamino]phenyl]methoxy]butanedioic 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 / L-glutamate import / response to decreased oxygen levels / cellular response to mercury ion / : / L-glutamate transmembrane transporter activity / retina layer formation / L-glutamate transmembrane transport / glutathione biosynthetic process / D-aspartate import across plasma membrane / L-aspartate transmembrane transport / cellular response to ammonium ion / righting reflex / zinc ion transmembrane transport / L-aspartate transmembrane transporter activity / cellular response to bisphenol A / intracellular glutamate homeostasis / grooming behavior / L-aspartate import across plasma membrane / Glutamate Neurotransmitter Release Cycle / monoatomic anion channel activity / L-glutamate import across plasma membrane / proximal dendrite / transepithelial transport / apical dendrite / intracellular zinc ion homeostasis / blood vessel morphogenesis / cellular response to cocaine / chloride transmembrane transporter activity / motor neuron apoptotic process / G protein-coupled dopamine receptor signaling pathway / response to anesthetic / response to morphine / glutamate receptor signaling pathway / superoxide metabolic process / neurotransmitter transport / heart contraction / perisynaptic space / maintenance of blood-brain barrier / dopamine metabolic process / adult behavior / motor behavior / asymmetric synapse / conditioned place preference / glial cell projection / synaptic cleft / behavioral fear response / postsynaptic modulation of chemical synaptic transmission / response to axon injury / positive regulation of heart rate / transport across blood-brain barrier / monoatomic ion transport / neurogenesis / axon terminus / chloride transmembrane transport / response to amphetamine / dendritic shaft / cell periphery / locomotory behavior / synapse organization / brain development / Schaffer collateral - CA1 synapse / memory / long-term synaptic potentiation / recycling endosome membrane / cytokine-mediated signaling pathway / late endosome membrane / presynapse / cellular response to oxidative stress / early endosome membrane / perikaryon / chemical synaptic transmission / gene expression / dendritic spine / negative regulation of neuron apoptotic process / apical plasma membrane / membrane raft / response to xenobiotic stimulus / axon / neuronal cell body / dendrite / cell surface / 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
Wellcome Trust106169/Z/14/Z United Kingdom
Biotechnology and Biological Sciences Research Council United Kingdom
CitationJournal: TO BE PUBLISHED
Title: Structure of human excitatory amino acid transporter 3 (EAAT3)
Authors: Baronina A / Pike ACW / Han S / Carpenter EP
History
DepositionJun 25, 2019-
Header (metadata) releaseJul 31, 2019-
Map releaseJul 8, 2020-
UpdateMay 22, 2024-
Current statusMay 22, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.035
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6s3q
  • Surface level: 0.035
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10094.png

Downloads & links

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Map

FileDownload / File: emd_10094.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPost-processed RELION map used for building and refinement
Projections & slices

Image control

Size
Brightness
Contrast
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

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.086 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.03 / Movie #1: 0.035
Minimum - Maximum-0.13491149 - 0.22698991
Average (Standard dev.)0.000032010787 (±0.007316282)
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-0.1350.2270.000

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Supplemental data

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Mask #1

Fileemd_10094_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: RELION 3D refine map

Fileemd_10094_additional.map
AnnotationRELION 3D refine map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION halfmap1

Fileemd_10094_half_map_1.map
AnnotationRELION halfmap1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: RELION halfmap2

Fileemd_10094_half_map_2.map
AnnotationRELION halfmap2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : EXCITATORY AMINO ACID TRANSPORTER 3 TRIMER

EntireName: EXCITATORY AMINO ACID TRANSPORTER 3 TRIMER
Components
  • Organelle or cellular component: EXCITATORY AMINO ACID TRANSPORTER 3 TRIMER
    • Protein or peptide: Excitatory amino acid transporter 3
  • Ligand: (2~{S},3~{S})-2-azanyl-3-[[3-[[4-(trifluoromethyl)phenyl]carbonylamino]phenyl]methoxy]butanedioic acid
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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Supramolecule #1: EXCITATORY AMINO ACID TRANSPORTER 3 TRIMER

SupramoleculeName: EXCITATORY AMINO ACID TRANSPORTER 3 TRIMER / type: organelle_or_cellular_component / 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: (2~{S},3~{S})-2-azanyl-3-[[3-[[4-(trifluoromethyl)phenyl]carbonyl...

MacromoleculeName: (2~{S},3~{S})-2-azanyl-3-[[3-[[4-(trifluoromethyl)phenyl]carbonylamino]phenyl]methoxy]butanedioic acid
type: ligand / ID: 2 / Number of copies: 3 / Formula: 7O9
Molecular weightTheoretical: 426.343 Da
Chemical component information

ChemComp-7O9:
(2~{S},3~{S})-2-azanyl-3-[[3-[[4-(trifluoromethyl)phenyl]carbonylamino]phenyl]methoxy]butanedioic acid

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Macromolecule #3: CHOLESTEROL HEMISUCCINATE

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

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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: 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
20.0 mMC8H18N2O4SHEPES
150.0 mMNaClsodium chloride
0.003 % (w/v)C47H88O22Lauryl Maltose Neopentyl Glycol
0.0003 % (w/v)C31H50O4Cholesteryl hemisuccinate
1.0 mMC19H17F3N2O6TFB-TBOA
0.01 % (w/v)C24H46O11dodecyl-beta-D-maltoside

Details: Protein incubated with 1mM TFB-TBOA solution prior to making grids
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: 278 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 4.5s.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-48 / Number grids imaged: 1 / Number real images: 3232 / Average exposure time: 12.0 sec. / Average electron dose: 40.7 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 281809
Details: Particles were picked based on 2D templates arising from classification of particles picked from a subset of images using gautomatch in non-template mode.
Startup modelType of model: OTHER
Details: Ab-initio reconstruction from selected particles in RELION3
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: 113258
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: Multiple rounds of 2D classification
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: C3 symmetry imposed
Final 3D classificationNumber classes: 10 / Avg.num./class: 13950 / Software - Name: RELION (ver. 3.0)
Details: Final best particles selected based on static classification using 10 classes after 3D refinement with no further image alignment
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6gct


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsModel refined using NCS constraints and rotamer, cbeta restraints only against RELION post-processed b-factor sharpened (-111A**2), 3.34A filtered map
RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 111 / Target criteria: Cross-correlation coefficient
Output model

PDB-6s3q:
Structure of human excitatory amino acid transporter 3 (EAAT3) in complex with TFB-TBOA

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