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- PDB-8afa: Cryo-EM structure of a substrate-bound glutamate transporter homo... -

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Basic information

Entry
Database: PDB / ID: 8afa
TitleCryo-EM structure of a substrate-bound glutamate transporter homologue GltTk encapsulated within a nanodisc
ComponentsProton/glutamate symporter, SDF family
KeywordsTRANSPORT PROTEIN / Glutamate / transporter / mutant / nanodisc
Function / homology
Function and homology information


carboxylic acid transport / symporter activity / membrane
Similarity search - Function
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
ASPARTIC ACID / Proton/glutamate symporter, SDF family
Similarity search - Component
Biological speciesThermococcus kodakarensis (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / Resolution: 3.16 Å
AuthorsWhittaker, J.J. / Guskov, A.
Funding support Netherlands, 1items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO) Netherlands
Citation
Journal: Nat Commun / Year: 2023
Title: Mutation in glutamate transporter homologue GltTk provides insights into pathologic mechanism of episodic ataxia 6.
Authors: Emanuela Colucci / Zaid R Anshari / Miyer F Patiño-Ruiz / Mariia Nemchinova / Jacob Whittaker / Dirk J Slotboom / Albert Guskov /
Abstract: Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an ...Episodic ataxias (EAs) are rare neurological conditions affecting the nervous system and typically leading to motor impairment. EA6 is linked to the mutation of a highly conserved proline into an arginine in the glutamate transporter EAAT1. In vitro studies showed that this mutation leads to a reduction in the substrates transport and an increase in the anion conductance. It was hypothesised that the structural basis of these opposed functional effects might be the straightening of transmembrane helix 5, which is kinked in the wild-type protein. In this study, we present the functional and structural implications of the mutation P208R in the archaeal homologue of glutamate transporters Glt. We show that also in Glt the P208R mutation leads to reduced aspartate transport activity and increased anion conductance, however a cryo-EM structure reveals that the kink is preserved. The arginine side chain of the mutant points towards the lipidic environment, where it may engage in interactions with the phospholipids, thereby potentially interfering with the transport cycle and contributing to stabilisation of an anion conducting state.
#1: Journal: Nat Commun / Year: 2020
Title: Structural ensemble of a glutamate transporter homologue in lipid nanodisc environment.
Authors: Valentina Arkhipova / Albert Guskov / Dirk J Slotboom /
Abstract: Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each ...Glutamate transporters are cation-coupled secondary active membrane transporters that clear the neurotransmitter L-glutamate from the synaptic cleft. These transporters are homotrimers, with each protomer functioning independently by an elevator-type mechanism, in which a mobile transport domain alternates between inward- and outward-oriented states. Using single-particle cryo-EM we have determined five structures of the glutamate transporter homologue Glt, a Na- L-aspartate symporter, embedded in lipid nanodiscs. Dependent on the substrate concentrations used, the protomers of the trimer adopt a variety of asymmetrical conformations, consistent with the independent movement. Six of the 15 resolved protomers are in a hitherto elusive state of the transport cycle in which the inward-facing transporters are loaded with Na ions. These structures explain how substrate-leakage is prevented - a strict requirement for coupled transport. The belt protein of the lipid nanodiscs bends around the inward oriented protomers, suggesting that membrane deformations occur during transport.
History
DepositionJul 16, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 5, 2023Provider: repository / Type: Initial release
Revision 1.1Apr 12, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Proton/glutamate symporter, SDF family
B: Proton/glutamate symporter, SDF family
C: Proton/glutamate symporter, SDF family
hetero molecules


Theoretical massNumber of molelcules
Total (without water)140,6366
Polymers140,2363
Non-polymers3993
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1(chain "C" and (resid 16 through 114 or resid 129 through 430 or resid 501))
d_2ens_1(chain "B" and (resid 16 through 114 or resid 129 through 430 or resid 501))
d_3ens_1(chain "A" and (resid 16 through 114 or resid 129 through 430 or resid 501))

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1TRPASNA16 - 114
d_12ens_1PROSERA129 - 430
d_13ens_1ASPASPD501
d_21ens_1TRPASNB16 - 114
d_22ens_1PROSERB129 - 430
d_23ens_1ASPASPE501
d_31ens_1TRPASNC16 - 114
d_32ens_1PROSERC129 - 430
d_33ens_1ASPASPF - B501

NCS oper:
IDCodeMatrixVector
1given(-0.504846269496, 0.863200726, -0.0038406782544), (-0.863208699805, -0.504835823899, 0.0033958046143), (0.000992349037528, 0.00502966617382, 0.999986858764)69.0970070346, 253.046168105, -0.736793879705
2given(-0.493140954074, -0.869915712403, 0.00765850699869), (0.869948759892, -0.493132341861, 0.00310621503735), (0.00107451222433, 0.00819431051322, 0.999965848766)252.055547024, 66.3017991837, -1.13176387129

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Components

#1: Protein Proton/glutamate symporter, SDF family


Mass: 46745.441 Da / Num. of mol.: 3 / Mutation: P208R
Source method: isolated from a genetically manipulated source
Details: Mutant P208R / Source: (gene. exp.) Thermococcus kodakarensis (archaea) / Strain: ATCC BAA-918 / JCM 12380 / KOD1 / Cell: E.coli / Gene: TK0986 / Production host: Thermococcus kodakarensis (archaea) / References: UniProt: Q5JID0
#2: Chemical ChemComp-ASP / ASPARTIC ACID / Aspartic acid


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C4H7NO4
Has ligand of interestN

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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: Glutamate transporter homologue GltTK in a lipid nanodisc environment bound with L-aspartate ligands
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 1.39 MDa / Experimental value: YES
Source (natural)Organism: Thermococcus kodakarensis (archaea) / Strain: BL21(DE3)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMtris(hydroxymethyl)aminomethaneC4H11NO31
2300 mMpotassium chlorideNaClSodium chloride1
30.01 mMDecyl maltosideC22H44O111
SpecimenConc.: 6.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: NO
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/4

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Electron microscopy imaging

MicroscopyModel: TFS TALOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: KODAK 4489 FILM
Image scansScanner model: TEMSCAN

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.16 Å / Resolution method: FSC 0.33 CUT-OFF / Num. of particles: 3000 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 50.94 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0089167
ELECTRON MICROSCOPYf_angle_d0.5912464
ELECTRON MICROSCOPYf_dihedral_angle_d2.6841280
ELECTRON MICROSCOPYf_chiral_restr0.0351561
ELECTRON MICROSCOPYf_plane_restr0.0051523
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2BELECTRON MICROSCOPYNCS constraints0.000706929875033
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.00070596921835

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