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- PDB-7nsg: Structure of human excitatory amino acid transporter 3 (EAAT3) in... -

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Basic information

Entry
Database: PDB / ID: 7nsg
TitleStructure of human excitatory amino acid transporter 3 (EAAT3) in complex with HIP-B
ComponentsExcitatory amino acid transporter 3
KeywordsMEMBRANE PROTEIN / GLUTAMATE / ASPARTATE TRANSPORTER / CYSTEINE / INHIBITOR COMPLEX / 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 / response to anesthetic / cysteine transmembrane transporter activity / cysteine transport / 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 / response to anesthetic / 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 / response to decreased oxygen levels / cellular response to mercury ion / Transport of inorganic cations/anions and amino acids/oligopeptides / retina layer formation / L-glutamate transmembrane transport / L-glutamate transmembrane transporter activity / zinc ion transmembrane transport / cellular response to bisphenol A / L-aspartate transmembrane transport / glutathione biosynthetic process / cellular response to ammonium ion / D-aspartate import across plasma membrane / righting reflex / apical dendrite / monoatomic anion channel activity / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / grooming behavior / intracellular glutamate homeostasis / Glutamate Neurotransmitter Release Cycle / proximal dendrite / L-glutamate import across plasma membrane / transepithelial transport / conditioned place preference / intracellular zinc ion homeostasis / cellular response to cocaine / blood vessel morphogenesis / neurotransmitter transport / motor behavior / response to morphine / motor neuron apoptotic process / chloride transmembrane transporter activity / glutamate binding / glutamate receptor signaling pathway / G protein-coupled dopamine receptor signaling pathway / positive regulation of heart rate / postsynaptic modulation of chemical synaptic transmission / maintenance of blood-brain barrier / superoxide metabolic process / heart contraction / adult behavior / perisynaptic space / dopamine metabolic process / cellular response to organic cyclic compound / glial cell projection / behavioral fear response / asymmetric synapse / transport across blood-brain barrier / response to axon injury / synaptic cleft / monoatomic ion transport / axon terminus / chloride transmembrane transport / response to amphetamine / neurogenesis / dendritic shaft / cell periphery / long-term synaptic potentiation / locomotory behavior / synapse organization / brain development / regulation of protein phosphorylation / Schaffer collateral - CA1 synapse / cytokine-mediated signaling pathway / memory / recycling endosome membrane / late endosome membrane / presynapse / cellular response to oxidative stress / gene expression / early endosome membrane / 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
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Chem-UR5 / Chem-UR8 / CHOLESTEROL HEMISUCCINATE / Excitatory amino acid transporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.34 Å
AuthorsBaronina, A. / Pike, A.C.W. / Yu, X. / Dong, Y.Y. / Shintre, C.A. / Tessitore, A. / Chu, A. / Rotty, B. / Venkaya, S. / Mukhopadhyay, S.M.M. ...Baronina, A. / Pike, A.C.W. / Yu, X. / Dong, Y.Y. / Shintre, C.A. / Tessitore, A. / Chu, A. / Rotty, B. / Venkaya, S. / Mukhopadhyay, S.M.M. / Borkowska, O. / Chalk, R. / Shrestha, L. / Burgess-Brown, N.A. / Edwards, A.M. / Arrowsmith, C.H. / Bountra, C. / Han, S. / Carpenter, E.P. / Structural Genomics Consortium (SGC)
Funding support United Kingdom, 2items
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, A.C.W. / Yu, X. / Dong, Y.Y. / Shintre, C.A. / Tessitore, A. / Chu, A. / Rotty, B. / Venkaya, S. / Mukhopadhyay, S.M.M. / Borkowska, O. / Chalk, R. / Shrestha, L. / ...Authors: Baronina, A. / Pike, A.C.W. / Yu, X. / Dong, Y.Y. / Shintre, C.A. / Tessitore, A. / Chu, A. / Rotty, B. / Venkaya, S. / Mukhopadhyay, S.M.M. / Borkowska, O. / Chalk, R. / Shrestha, L. / Burgess-Brown, N.A. / Edwards, A.M. / Arrowsmith, C.H. / Bountra, C. / Han, S. / Carpenter, E.P.
History
DepositionMar 5, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 16, 2022Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2024Group: Data collection / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / em_3d_fitting_list / em_admin / pdbx_initial_refinement_model
Item: _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id ..._em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update

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Structure visualization

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Assembly

Deposited unit
A: Excitatory amino acid transporter 3
B: Excitatory amino acid transporter 3
C: Excitatory amino acid transporter 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)174,10427
Polymers159,7593
Non-polymers14,34524
Water00
1


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area19400 Å2
ΔGint-193 kcal/mol
Surface area56200 Å2
MethodPISA

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Components

#1: Protein Excitatory amino acid transporter 3 / Excitatory amino-acid carrier 1 / Neuronal and epithelial glutamate transporter / Sodium-dependent ...Excitatory amino-acid carrier 1 / Neuronal and epithelial glutamate transporter / Sodium-dependent glutamate/aspartate transporter 3 / Solute carrier family 1 member 1


Mass: 53252.867 Da / Num. of mol.: 3 / Mutation: S42R, L92V
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC1A1, EAAC1, EAAT3 / Production host: Spodoptera frugiperda (fall armyworm) / Strain (production host): 9 / References: UniProt: P43005
#2: Chemical ChemComp-UR5 / (+)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid / (3~{a}~{S},6~{S},6~{a}~{S})-3-oxidanylidene-4,5,6,6~{a}-tetrahydro-3~{a}~{H}-pyrrolo[3,4-d][1,2]oxazole-6-carboxylic acid


Mass: 172.139 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H8N2O4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-UR8 / (-)-3-Hydroxy-4,5,6,6a-tetrahydro-3aH-pyrrolo[3,4-d]isoxazole-6-carboxylic acid / (3~{a}~{R},6~{R},6~{a}~{R})-3-oxidanylidene-4,5,6,6~{a}-tetrahydro-3~{a}~{H}-pyrrolo[3,4-d][1,2]oxazole-6-carboxylic acid


Mass: 172.139 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C6H8N2O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C31H50O4
#5: Chemical
ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE


Mass: 790.145 Da / Num. of mol.: 15 / 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: Ternary complex of excitatory amino acid transporter 3 with inhibitor HIP-B
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.16 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Strain: 9
Buffer solutionpH: 7.5
Details: Solutions were made fresh from pre-made, sterile filtered stocks.
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
220 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
30.003 %lauryl maltose neopentyl glycolLMNG1
40.0003 %cholesteryl hemisuccinateCHS1
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample after SEC
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: Blot time 4s Blot force -15 sample volume 3 uL

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1600 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 42 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 4947
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategoryDetails
1Gautomatch0.53particle selectionTemplate based picking using Gautomatch
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7Coot0.8.9model fitting
9PHENIX1.14model refinement
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1169680
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.34 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 127936 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Details: MODEL 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
Atomic model buildingPDB-ID: 6S3Q

6s3q


Pdb chain-ID: A / Accession code: 6S3Q / Source name: PDB / Type: experimental model
RefinementStereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 49.47 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.007610497
ELECTRON MICROSCOPYf_angle_d0.882214175
ELECTRON MICROSCOPYf_chiral_restr0.05091761
ELECTRON MICROSCOPYf_plane_restr0.00681692
ELECTRON MICROSCOPYf_dihedral_angle_d14.88054035

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