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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 / response to anesthetic / regulation of protein targeting to membrane / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / response to decreased oxygen levels / distal dendrite / cysteine transmembrane transporter activity / cysteine transport / high-affinity L-glutamate transmembrane transporter activity ...D-aspartate transmembrane transport / response to anesthetic / regulation of protein targeting to membrane / D-aspartate transmembrane transporter activity / Defective SLC1A1 is implicated in schizophrenia 18 (SCZD18) and dicarboxylic aminoaciduria (DCBXA) / response to decreased oxygen levels / distal dendrite / cysteine transmembrane transporter activity / cysteine transport / high-affinity L-glutamate transmembrane transporter activity / glutamate:sodium symporter activity / L-glutamate import / neurotransmitter receptor transport to plasma membrane / cellular response to mercury ion / Transport of inorganic cations/anions and amino acids/oligopeptides / zinc ion transmembrane transport / retina layer formation / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / monoatomic anion channel activity / cellular response to bisphenol A / L-aspartate transmembrane transport / D-aspartate import across plasma membrane / cellular response to ammonium ion / proximal dendrite / glutathione biosynthetic process / righting reflex / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / grooming behavior / intracellular glutamate homeostasis / Glutamate Neurotransmitter Release Cycle / L-glutamate import across plasma membrane / conditioned place preference / transepithelial transport / apical dendrite / intracellular zinc ion homeostasis / glutamate receptor signaling pathway / response to morphine / neurotransmitter transport / cellular response to cocaine / blood vessel morphogenesis / motor behavior / motor neuron apoptotic process / glutamate binding / chloride transmembrane transporter activity / G protein-coupled dopamine receptor signaling pathway / adult behavior / positive regulation of heart rate / postsynaptic modulation of chemical synaptic transmission / maintenance of blood-brain barrier / dopamine metabolic process / superoxide metabolic process / heart contraction / perisynaptic space / glial cell projection / cellular response to organic cyclic compound / transport across blood-brain barrier / asymmetric synapse / response to axon injury / behavioral fear response / monoatomic ion transport / synaptic cleft / axon terminus / chloride transmembrane transport / response to amphetamine / neurogenesis / dendritic shaft / locomotory behavior / cell periphery / long-term synaptic potentiation / synapse organization / regulation of protein phosphorylation / Schaffer collateral - CA1 synapse / brain development / memory / cytokine-mediated signaling pathway / recycling endosome membrane / presynapse / late endosome membrane / gene expression / early endosome membrane / cellular response to oxidative stress / chemical synaptic transmission / perikaryon / negative regulation of neuron apoptotic process / dendritic spine / response to xenobiotic stimulus / membrane raft / apical plasma membrane / axon / neuronal cell body / dendrite / cell surface / endoplasmic reticulum / extracellular exosome / identical protein binding / 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 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
Voxel sizeX=Y=Z: 1.086 Å
Density
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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