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- EMDB-24405: GltPh mutant (S279E/D405N) in complex with aspartate and sodium ions -

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Basic information

Entry
Database: EMDB / ID: EMD-24405
TitleGltPh mutant (S279E/D405N) in complex with aspartate and sodium ions
Map data20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A
Sample
  • Organelle or cellular component: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and aspartate
    • Protein or peptide: Glutamate transporter homolog
  • Ligand: ASPARTIC ACID
  • Ligand: SODIUM ION
  • Ligand: water
Keywordsoutward-facing / substrate-bound / TRANSPORT PROTEIN
Function / homology
Function and homology information


amino acid:sodium symporter activity / L-aspartate transmembrane transport / L-aspartate transmembrane transporter activity / L-aspartate import across plasma membrane / chloride transmembrane transporter activity / protein homotrimerization / chloride transmembrane transport / identical protein binding / metal ion binding / plasma 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
Glutamate transporter homolog
Similarity search - Component
Biological speciesPyrococcus horikoshii (archaea) / Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsReddy KD / Boudker O
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS064357 United States
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)NS102325 United States
CitationJournal: J Gen Physiol / Year: 2022
Title: The archaeal glutamate transporter homologue GltPh shows heterogeneous substrate binding.
Authors: Krishna D Reddy / Didar Ciftci / Amanda J Scopelliti / Olga Boudker /
Abstract: Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent ...Integral membrane glutamate transporters couple the concentrative substrate transport to ion gradients. There is a wealth of structural and mechanistic information about this protein family. Recent studies of an archaeal homologue, GltPh, revealed transport rate heterogeneity, which is inconsistent with simple kinetic models; however, its structural and mechanistic determinants remain undefined. Here, we demonstrate that in a mutant GltPh, which exclusively populates the outward-facing state, at least two substates coexist in slow equilibrium, binding the substrate with different apparent affinities. Wild type GltPh shows similar binding properties, and modulation of the substate equilibrium correlates with transport rates. The low-affinity substate of the mutant is transient following substrate binding. Consistently, cryo-EM on samples frozen within seconds after substrate addition reveals the presence of structural classes with perturbed helical packing of the extracellular half of the transport domain in regions adjacent to the binding site. By contrast, an equilibrated structure does not show such classes. The structure at 2.2-Å resolution details a pattern of waters in the intracellular half of the domain and resolves classes with subtle differences in the substrate-binding site. We hypothesize that the rigid cytoplasmic half of the domain mediates substrate and ion recognition and coupling, whereas the extracellular labile half sets the affinity and dynamic properties.
History
DepositionJul 7, 2021-
Header (metadata) releaseApr 20, 2022-
Map releaseApr 20, 2022-
UpdateJun 5, 2024-
Current statusJun 5, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24405.png

Downloads & links

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Map

FileDownload / File: emd_24405.map.gz / Format: CCP4 / Size: 343 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A
Voxel sizeX=Y=Z: 0.81156 Å
Density
Contour LevelBy AUTHOR: 2.43
Minimum - Maximum-10.440334999999999 - 19.858042000000001
Average (Standard dev.)0.000024037343 (±0.24284479)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions448448448
Spacing448448448
CellA=B=C: 363.58 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...

Fileemd_24405_additional_1.map
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A, Class A2-4
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...

Fileemd_24405_additional_2.map
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A, Class A2-1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...

Fileemd_24405_additional_3.map
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A, Class A1-4
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...

Fileemd_24405_additional_4.map
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A, Class A1-8
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of glutamate transporter homologue GltPh mutant (S279E/D4...

EntireName: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and aspartate
Components
  • Organelle or cellular component: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and aspartate
    • Protein or peptide: Glutamate transporter homolog
  • Ligand: ASPARTIC ACID
  • Ligand: SODIUM ION
  • Ligand: water

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Supramolecule #1: Complex of glutamate transporter homologue GltPh mutant (S279E/D4...

SupramoleculeName: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and aspartate
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pyrococcus horikoshii (archaea)

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Macromolecule #1: Glutamate transporter homolog

MacromoleculeName: Glutamate transporter homolog / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus horikoshii (strain ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3) (archaea)
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Molecular weightTheoretical: 43.927055 KDa
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. DH10B (bacteria)
SequenceString: GLYRKYIEYP VLQKILIGLI LGAIVGLILG HYGYAHAVHT YVKPFGDLFV RLLKMLVMPI VFASLVVGAA SISPARLGRV GVKIVVYYL LTSAFAVTLG IIMARLFNPG AGIHLAVGGQ QFQPHQAPPL VHILLDIVPT NPFGALANGQ VLPTIFFAII L GIAITYLM ...String:
GLYRKYIEYP VLQKILIGLI LGAIVGLILG HYGYAHAVHT YVKPFGDLFV RLLKMLVMPI VFASLVVGAA SISPARLGRV GVKIVVYYL LTSAFAVTLG IIMARLFNPG AGIHLAVGGQ QFQPHQAPPL VHILLDIVPT NPFGALANGQ VLPTIFFAII L GIAITYLM NSENEKVRKS AETLLDAING LAEAMYKIVN GVMQYAPIGV FALIAYVMAE QGVHVVGELA KVTAAVYVGL TL QILLVYF VLLKIYGIDP ISFIKHAKDA MLTAFVTRSS EGTLPVTMRV AKEMGISEGI YSFTLPLGAT INMDGTALYQ GVC TFFIAN ALGSHLTVGQ QLTIVLTAVL ASIGTAGVPG AGAIMLAMVL HSVGLPLTDP NVAAAYAMIL GIDAILDMGR TMVN VTGNL TGTAIVAKTE

UniProtKB: Glutamate transporter homolog

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Macromolecule #2: ASPARTIC ACID

MacromoleculeName: ASPARTIC ACID / type: ligand / ID: 2 / Number of copies: 3 / Formula: ASP
Molecular weightTheoretical: 133.103 Da
Chemical component information

ChemComp-ASP:
ASPARTIC ACID

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Macromolecule #3: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 3 / Number of copies: 6
Molecular weightTheoretical: 22.99 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 57 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

BufferpH: 7.4
Component:
ConcentrationName
20.0 mMHEPES
250.0 mMsodium nitrate
0.8 mMDDM
1.0 mML-Asp
Sugar embeddingMaterial: ice
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.91 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 503427
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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