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- PDB-7ugv: Asp-bound GltPh RSMR mutant in IFS-A2 state -

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Basic information

Entry
Database: PDB / ID: 7ugv
TitleAsp-bound GltPh RSMR mutant in IFS-A2 state
ComponentsGlutamate transporter homolog
KeywordsTRANSPORT PROTEIN / GltPh / Inward-facing state / glutamate transporter
Function / homology
Function and homology information


L-aspartate transmembrane transport / amino acid:sodium symporter activity / L-aspartate transmembrane transporter 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
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: 2.94 Å
AuthorsHuang, Y. / Boudker, O.
Funding support United States, 2items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS085318 United States
CitationJournal: J Am Chem Soc / Year: 2023
Title: Environmentally Ultrasensitive Fluorine Probe to Resolve Protein Conformational Ensembles by F NMR and Cryo-EM.
Authors: Yun Huang / Krishna D Reddy / Clay Bracken / Biao Qiu / Wenhu Zhan / David Eliezer / Olga Boudker /
Abstract: Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by F NMR. We describe a novel monofluoroethyl F probe that dramatically ...Limited chemical shift dispersion represents a significant barrier to studying multistate equilibria of large membrane proteins by F NMR. We describe a novel monofluoroethyl F probe that dramatically increases the chemical shift dispersion. The improved conformational sensitivity and line shape enable the detection of previously unresolved states in one-dimensional (1D) F NMR spectra of a 134 kDa membrane transporter. Changes in the populations of these states in response to ligand binding, mutations, and temperature correlate with population changes of distinct conformations in structural ensembles determined by single-particle cryo-electron microscopy (cryo-EM). Thus, F NMR can guide sample preparation to discover and visualize novel conformational states and facilitate image analysis and three-dimensional (3D) classification.
History
DepositionMar 25, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release
Revision 1.1May 10, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _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

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Glutamate transporter homolog
hetero molecules


Theoretical massNumber of molelcules
Total (without water)44,3355
Polymers44,1331
Non-polymers2024
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 44133.230 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Pyrococcus horikoshii (archaea)
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Gene: PH1295 / Production host: Escherichia coli (E. coli) / References: UniProt: O59010
#2: Chemical ChemComp-ASP / ASPARTIC ACID


Type: L-peptide linking / Mass: 133.103 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C4H7NO4 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-NA / SODIUM ION


Mass: 22.990 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Na / 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: GltPh / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Pyrococcus horikoshii (archaea)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4
SpecimenConc.: 4.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 303 K

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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 / Nominal defocus max: 1500 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 50.94 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
7UCSF Chimera1.14model fitting
13PHENIX1.19.2-4158-000model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 335559 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT
Atomic model buildingPDB-ID: 3KBC

3kbc


Pdb chain-ID: A
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023165
ELECTRON MICROSCOPYf_angle_d0.4564315
ELECTRON MICROSCOPYf_dihedral_angle_d4.124445
ELECTRON MICROSCOPYf_chiral_restr0.036540
ELECTRON MICROSCOPYf_plane_restr0.004532

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