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- PDB-7rcp: GltPh mutant (S279E/D405N) in complex with aspartate and sodium ions -

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Basic information

Entry
Database: PDB / ID: 7rcp
TitleGltPh mutant (S279E/D405N) in complex with aspartate and sodium ions
ComponentsGlutamate transporter homolog
KeywordsTRANSPORT PROTEIN / outward-facing / substrate-bound
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
ASPARTIC ACID / Glutamate transporter homolog
Similarity search - Component
Biological speciesPyrococcus horikoshii (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsReddy, K.D. / Boudker, O.
Funding support United States, 3items
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, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 20, 2022Provider: repository / Type: Initial release
Revision 1.1Nov 2, 2022Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Glutamate transporter homolog
B: Glutamate transporter homolog
C: Glutamate transporter homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)132,31812
Polymers131,7813
Non-polymers5379
Water1,02757
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_1chain "B"
d_2ens_1chain "A"
d_3ens_1chain "C"

NCS domain segments:
Dom-IDComponent-IDEns-IDBeg label comp-IDEnd label comp-IDLabel asym-IDLabel seq-ID
d_11ens_1GLYGLUB1 - 415
d_12ens_1ASPASPG - B1
d_13ens_1NANAB
d_21ens_1GLYGLUA1 - 415
d_22ens_1ASPASPA1
d_23ens_1NANAA
d_31ens_1GLYGLUC1 - 415
d_32ens_1ASPASPC1
d_33ens_1NANAC

NCS oper:
IDCodeMatrixVector
1given(-0.500145314473, 0.865941462529, -0.000218822308181), (-0.865941444886, -0.500145354075, -0.000197040832326), (-0.000280068787327, 9.09382566717E-5, 0.999999956646)115.328144333, 430.145586005, 0.0335334200561
2given(-0.499789740052, -0.866146721937, -0.000267986367577), (0.866146743887, -0.49978977054, 57600903064), (-0.000183827678529, -0.000203327179314, 0.999999962433)430.137471999, 115.181535141, 0.0689484860879

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Components

#1: Protein Glutamate transporter homolog / Glt(Ph) / Sodium-aspartate symporter Glt(Ph) / Sodium-dependent aspartate transporter


Mass: 43927.055 Da / Num. of mol.: 3 / Mutation: S279E, D405N
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) 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
Gene: PH1295
Production host: Escherichia coli str. K-12 substr. DH10B (bacteria)
References: UniProt: O59010
#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 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Na / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 57 / Source method: isolated from a natural source / Formula: H2O
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 glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and aspartate
Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Pyrococcus horikoshii (archaea)
Source (recombinant)Organism: Escherichia coli str. K-12 substr. DH10B (bacteria)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
120 mMHEPES1
2250 mMsodium nitrate1
30.8 mMDDM1
41 mML-Asp1
SpecimenEmbedding applied: YES / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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: 47.91 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.19.2_4158refinement
PHENIX1.19.2_4158refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 503427 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 33.78 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00489456
ELECTRON MICROSCOPYf_angle_d0.619512894
ELECTRON MICROSCOPYf_chiral_restr0.04581614
ELECTRON MICROSCOPYf_plane_restr0.00491590
ELECTRON MICROSCOPYf_dihedral_angle_d3.34651323
Refine LS restraints NCS
Ens-IDDom-IDAuth asym-IDRefine-IDTypeRms dev position (Å)
ens_1d_2AELECTRON MICROSCOPYNCS constraints0.0658300768702
ens_1d_3CELECTRON MICROSCOPYNCS constraints0.0658317068214

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