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- EMDB-26497: Asp-bound GltPh RSMR mutant in IFS-A2 state -

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Basic information

Entry
Database: EMDB / ID: EMD-26497
TitleAsp-bound GltPh RSMR mutant in IFS-A2 state
Map dataAsp-bound GltPh RSMR mutant in IFS state
Sample
  • Complex: GltPh
    • Protein or peptide: Glutamate transporter homolog
  • Ligand: ASPARTIC ACID
  • Ligand: SODIUM ION
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 Å
AuthorsHuang Y / Boudker O
Funding support United States, 2 items
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, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateMay 10, 2023-
Current statusMay 10, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26497.png

Downloads & links

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Map

FileDownload / File: emd_26497.map.gz / Format: CCP4 / Size: 166.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationAsp-bound GltPh RSMR mutant in IFS state
Voxel sizeX=Y=Z: 0.852 Å
Density
Contour LevelBy AUTHOR: 0.488
Minimum - Maximum-0.80923104 - 1.7369995
Average (Standard dev.)0.0025361392 (±0.03558632)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions352352352
Spacing352352352
CellA=B=C: 299.904 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half Map 1

Fileemd_26497_half_map_1.map
AnnotationHalf Map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half Map 2

Fileemd_26497_half_map_2.map
AnnotationHalf Map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : GltPh

EntireName: GltPh
Components
  • Complex: GltPh
    • Protein or peptide: Glutamate transporter homolog
  • Ligand: ASPARTIC ACID
  • Ligand: SODIUM ION

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Supramolecule #1: GltPh

SupramoleculeName: GltPh / type: complex / ID: 1 / Chimera: Yes / 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: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus horikoshii (archaea)
Strain: ATCC 700860 / DSM 12428 / JCM 9974 / NBRC 100139 / OT-3
Molecular weightTheoretical: 44.13323 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGLYRKYIEY PVLQKILIGL ILGAIVGLIL GHYGYAHAVH TYVKPFGDLF VRLLKMLVMP IVFASLVVGA ASISPARLGR VGVKIVVYY LLTSAFAVTL GIIMARLFNP GAGIHLAVGG QQFQPHQAPP LVHILLDIVP TNPFGALANG QVLPTIFFAI I LGIAITYL ...String:
MGLYRKYIEY PVLQKILIGL ILGAIVGLIL GHYGYAHAVH TYVKPFGDLF VRLLKMLVMP IVFASLVVGA ASISPARLGR VGVKIVVYY LLTSAFAVTL GIIMARLFNP GAGIHLAVGG QQFQPHQAPP LVHILLDIVP TNPFGALANG QVLPTIFFAI I LGIAITYL MNSENEKVRK SAETLLDAIN GLAEAMYKIV NGVMQYAPIG VFALIAHVMA HQGVHVVGEL AKVTAAVYVG LT LQILLVY FVLLKIYGID PISFIKHAKD AMLTAFVTSS SSGTLPVTMR VAKEMGISEG IYSFTLPLGA TINMDGTALY QGV ATFFIA NALGSHLTVG QQLTIVLTAV LASIGTAGVP GAGAIMLAMV LHSVGLPLTD PNVAAAYA(EFC)I LGIDAILDRG RTMVNVTGD LTGTAIVAKT EGT

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Macromolecule #2: ASPARTIC ACID

MacromoleculeName: ASPARTIC ACID / type: ligand / ID: 2 / Number of copies: 1 / 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: 3
Molecular weightTheoretical: 22.99 Da

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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

Concentration4.5 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 303 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.94 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 1.3 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3kbc

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 335559
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

3kbc


Chain - Chain ID: A
RefinementProtocol: FLEXIBLE FIT
Output model

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

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