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- EMDB-24405: GltPh mutant (S279E/D405N) in complex with aspartate and sodium ions -
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Open data
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Basic information
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Title | GltPh mutant (S279E/D405N) in complex with aspartate and sodium ions | ||||||||||||
![]() | 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A | ||||||||||||
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![]() | outward-facing / substrate-bound / TRANSPORT PROTEIN | ||||||||||||
Function / homology | ![]() 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 | ||||||||||||
Biological species | ![]() ![]() ![]() ![]() | ||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.2 Å | ||||||||||||
![]() | Reddy KD / Boudker O | ||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 19.2 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 20.4 KB 20.4 KB | Display Display | ![]() |
Images | ![]() | 118.9 KB | ||
Filedesc metadata | ![]() | 5.7 KB | ||
Others | ![]() ![]() ![]() ![]() | 21 MB 21 MB 21 MB 21 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7rcpMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Related items in Molecule of the Month |
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Map
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Annotation | 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.81156 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...
File | emd_24405_additional_1.map | ||||||||||||
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Annotation | 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A, Class A2-4 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...
File | emd_24405_additional_2.map | ||||||||||||
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Annotation | 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A, Class A2-1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...
File | emd_24405_additional_3.map | ||||||||||||
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Annotation | 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A, Class A1-4 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...
File | emd_24405_additional_4.map | ||||||||||||
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Annotation | 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset A, Class A1-8 | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : Complex of glutamate transporter homologue GltPh mutant (S279E/D4...
Entire | Name: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and aspartate |
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Components |
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-Supramolecule #1: Complex of glutamate transporter homologue GltPh mutant (S279E/D4...
Supramolecule | Name: 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 |
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Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: Glutamate transporter homolog
Macromolecule | Name: Glutamate transporter homolog / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() ![]() Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3 |
Molecular weight | Theoretical: 43.927055 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: ASPARTIC ACID
Macromolecule | Name: ASPARTIC ACID / type: ligand / ID: 2 / Number of copies: 3 / Formula: ASP |
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Molecular weight | Theoretical: 133.103 Da |
Chemical component information | ![]() ChemComp-ASP: |
-Macromolecule #3: SODIUM ION
Macromolecule | Name: SODIUM ION / type: ligand / ID: 3 / Number of copies: 6 |
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Molecular weight | Theoretical: 22.99 Da |
-Macromolecule #4: water
Macromolecule | Name: water / type: ligand / ID: 4 / Number of copies: 57 / Formula: HOH |
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Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ![]() ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.4 Component:
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Sugar embedding | Material: ice | ||||||||||
Vitrification | Cryogen name: ETHANE |
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Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 47.91 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
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Image processing
Startup model | Type of model: PDB ENTRY |
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Final reconstruction | Applied 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 assignment | Type: ANGULAR RECONSTITUTION |
Final angle assignment | Type: ANGULAR RECONSTITUTION |