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- PDB-6uwl: GltPh in complex with L-aspartate and sodium ions in intermediate... -

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Basic information

Entry
Database: PDB / ID: 6uwl
TitleGltPh in complex with L-aspartate and sodium ions in intermediate outward-facing state
ComponentsGlutamate transporter homolog
KeywordsTRANSPORT PROTEIN / aspartate transporter
Function / homology
Function and homology information


L-aspartate transmembrane transport / L-aspartate transmembrane transporter activity / amino acid:sodium symporter activity / L-aspartate import across plasma membrane / chloride transmembrane transporter activity / protein homotrimerization / chloride transmembrane transport / metal ion binding / identical protein binding / plasma membrane
Similarity search - Function
Proton glutamate symport protein / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter / Sodium:dicarboxylate symporter, conserved site / Sodium:dicarboxylate symporter superfamily / Sodium:dicarboxylate symporter family / Sodium:dicarboxylate symporter family signature 1. / Orthogonal Bundle / Mainly Alpha
Similarity search - Domain/homology
Chem-6OU / ASPARTIC ACID / Glutamate transporter homolog
Similarity search - Component
Biological speciesPyrococcus horikoshii (archaea)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.62 Å
AuthorsWang, X. / Boudker, O.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R37NS085318 United States
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)R01NS064357 United States
CitationJournal: Nat Chem Biol / Year: 2020
Title: Use of paramagnetic F NMR to monitor domain movement in a glutamate transporter homolog.
Authors: Yun Huang / Xiaoyu Wang / Guohua Lv / Asghar M Razavi / Gerard H M Huysmans / Harel Weinstein / Clay Bracken / David Eliezer / Olga Boudker /
Abstract: In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel F-NMR spectroscopy ...In proteins where conformational changes are functionally important, the number of accessible states and their dynamics are often difficult to establish. Here we describe a novel F-NMR spectroscopy approach to probe dynamics of large membrane proteins. We labeled a glutamate transporter homolog with a F probe via cysteine chemistry and with a Ni ion via chelation by a di-histidine motif. We used distance-dependent enhancement of the longitudinal relaxation of F nuclei by the paramagnetic metal to assign the observed resonances. We identified one inward- and two outward-facing states of the transporter, in which the substrate-binding site is near the extracellular and intracellular solutions, respectively. We then resolved the structure of the unanticipated second outward-facing state by cryo-EM. Finally, we showed that the rates of the conformational exchange are accessible from measurements of the metal-enhanced longitudinal relaxation of F nuclei.
History
DepositionNov 5, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 22, 2020Provider: repository / Type: Initial release
Revision 1.1Nov 18, 2020Group: 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.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.2Dec 25, 2024Group: Advisory / Data collection ...Advisory / Data collection / Database references / Derived calculations / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature / pdbx_unobs_or_zero_occ_atoms / pdbx_validate_close_contact / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Assembly

Deposited unit
A: Glutamate transporter homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)45,5213
Polymers44,6701
Non-polymers8512
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area0 Å2
ΔGint0 kcal/mol
Surface area18330 Å2

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Components

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


Mass: 44669.957 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus horikoshii (archaea) / Production host: Escherichia coli (E. coli) / References: UniProt: O59010*PLUS
#2: Chemical ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H7NO4
#3: 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.: 1 / Source method: obtained synthetically / Formula: C39H76NO8P / Comment: phospholipid*YM
Has ligand of interestN
Has protein modificationY

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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 GltPh with L-aspartate and sodium ions 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
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 FIELD
Image recording
IDImaging-IDElectron dose (e/Å2)Film or detector model
1150.1615GATAN K3 (6k x 4k)
2150.1615GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: (1.14_3260: phenix.real_space_refine) / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.62 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120280 / Symmetry type: POINT

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