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- PDB-6x16: Inward-facing state of the glutamate transporter homologue GltPh ... -

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Entry
Database: PDB / ID: 6x16
TitleInward-facing state of the glutamate transporter homologue GltPh in complex with TBOA
ComponentsGlutamate transporter homologue GltPh
KeywordsTRANSPORT PROTEIN / sodium-coupled L-aspartate transporter
Biological speciesPyrococcus horikoshii (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsWang, X. / Boudker, O.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS085318 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS064357 United States
CitationJournal: Elife / Year: 2020
Title: Large domain movements through the lipid bilayer mediate substrate release and inhibition of glutamate transporters.
Authors: Xiaoyu Wang / Olga Boudker /
Abstract: Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. ...Glutamate transporters are essential players in glutamatergic neurotransmission in the brain, where they maintain extracellular glutamate below cytotoxic levels and allow for rounds of transmission. The structural bases of their function are well established, particularly within a model archaeal homolog, sodium, and aspartate symporter Glt. However, the mechanism of gating on the cytoplasmic side of the membrane remains ambiguous. We report Cryo-EM structures of Glt reconstituted into nanodiscs, including those structurally constrained in the cytoplasm-facing state and either apo, bound to sodium ions only, substrate, or blockers. The structures show that both substrate translocation and release involve movements of the bulky transport domain through the lipid bilayer. They further reveal a novel mode of inhibitor binding and show how solutes release is coupled to protein conformational changes. Finally, we describe how domain movements are associated with the displacement of bound lipids and significant membrane deformations, highlighting the potential regulatory role of the bilayer.
Validation Report
SummaryFull reportAbout validation report
History
DepositionMay 18, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 18, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 25, 2020Group: Data processing / Category: em_3d_reconstruction / Item: _em_3d_reconstruction.resolution

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Assembly

Deposited unit
B: Glutamate transporter homologue GltPh
A: Glutamate transporter homologue GltPh
C: Glutamate transporter homologue GltPh
hetero molecules


Theoretical massNumber of molelcules
Total (without water)136,8039
Polymers133,9323
Non-polymers2,8726
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area9150 Å2
ΔGint-47 kcal/mol
Surface area48640 Å2

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Components

#1: Protein Glutamate transporter homologue GltPh


Mass: 44643.918 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus horikoshii (archaea) / Production host: Escherichia coli (E. coli)
#2: Chemical ChemComp-6OU / [(2~{R})-1-[2-azanylethoxy(oxidanyl)phosphoryl]oxy-3-hexadecanoyloxy-propan-2-yl] (~{Z})-octadec-9-enoate


Mass: 717.996 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C39H76NO8P / Comment: phospholipid*YM
#3: Chemical ChemComp-TB1 / (3S)-3-(BENZYLOXY)-L-ASPARTIC ACID / D,L-THREO-BENZYLOXYASPARTATE


Mass: 239.225 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C11H13NO5 / Feature type: SUBJECT OF INVESTIGATION
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: Complex of inward-facing state of GltPh with TBOA in MSP1E3 nanodisc
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.134 MDa / Experimental value: NO
Source (natural)Organism: Pyrococcus horikoshii (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
EM embeddingMaterial: ice
VitrificationCryogen name: ETHANE

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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 FIELDBright-field microscopy
Image recordingElectron dose: 69.7 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 11035 / Symmetry type: POINT

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