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- EMDB-24406: Glutamate transporter homologue GltPh mutant - S279E/D405N -

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Basic information

Entry
Database: EMDB / ID: EMD-24406
TitleGlutamate transporter homologue GltPh mutant - S279E/D405N
Map data20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset B, Class B3 - substrate added just prior to freezing
Sample
  • Organelle or cellular component: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and fast addition of aspartate
    • Protein or peptide: Glutamate transporter homologue GltPh mutant - S279E/D405N
Biological speciesPyrococcus horikoshii (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.0 Å
AuthorsReddy KD / Boudker O
Funding support United States, 3 items
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: To Be Published
Title: Heterogeneous substrate binding of a glutamate transporter homologue
Authors: Reddy KD / Ciftci D / Scopelliti A / Boudker O
History
DepositionJul 7, 2021-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateJul 13, 2022-
Current statusJul 13, 2022Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_24406.png

Downloads & links

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Map

FileDownload / File: emd_24406.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset B, Class B3 - substrate added just prior to freezing
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.07 Å/pix.
x 240 pix.
= 257.52 Å		1.07 Å/pix.
x 240 pix.
= 257.52 Å		1.07 Å/pix.
x 240 pix.
= 257.52 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.073 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 2.83
Minimum - Maximum-11.737695 - 21.271355
Average (Standard dev.)4.5563433e-05 (±0.47169235)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 257.52 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...

Fileemd_24406_additional_1.map
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset B, Class B1 - substrate added just prior to freezing
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...

Fileemd_24406_additional_2.map
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset B, Class B6 - substrate added just prior to freezing
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: 20 mM HEPES/Tris pH 7.4, 250 mM NaNO3,...

Fileemd_24406_additional_3.map
Annotation20 mM HEPES/Tris pH 7.4, 250 mM NaNO3, 1 mM L-Asp Dataset B, Class B7 - substrate added just prior to freezing
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of glutamate transporter homologue GltPh mutant (S279E/D4...

EntireName: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and fast addition of aspartate
Components
  • Organelle or cellular component: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and fast addition of aspartate
    • Protein or peptide: Glutamate transporter homologue GltPh mutant - S279E/D405N

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Supramolecule #1: Complex of glutamate transporter homologue GltPh mutant (S279E/D4...

SupramoleculeName: Complex of glutamate transporter homologue GltPh mutant (S279E/D405N) with sodium nitrate and fast addition of aspartate
type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Protein was purified in the presence of sodium only (250 mM NaNO3). Aspartate was added just prior to grid freezing (~5s).
Source (natural)Organism: Pyrococcus horikoshii (archaea)
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. DH10B (bacteria)

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Macromolecule #1: Glutamate transporter homologue GltPh mutant - S279E/D405N

MacromoleculeName: Glutamate transporter homologue GltPh mutant - S279E/D405N
type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Pyrococcus horikoshii (archaea)
Recombinant expressionOrganism: Escherichia coli str. K-12 substr. DH10B (bacteria)
SequenceString: GLYRKYIEYP VLQKILIGLI LGAIVGLILG HYGYAHAVHT YVKPFGDLFV RLLKMLVMPI VFASLVVGAA SISPARLGRV GVKIVVYYLL TSAFAVTLGI IMARLFNPGA GIHLAVGGQQ FQPHQAPPLV HILLDIVPTN PFGALANGQV LPTIFFAIIL GIAITYLMNS ...String:
GLYRKYIEYP VLQKILIGLI LGAIVGLILG HYGYAHAVHT YVKPFGDLFV RLLKMLVMPI VFASLVVGAA SISPARLGRV GVKIVVYYLL TSAFAVTLGI IMARLFNPGA GIHLAVGGQQ FQPHQAPPLV HILLDIVPTN PFGALANGQV LPTIFFAIIL GIAITYLMNS ENEKVRKSAE TLLDAINGLA EAMYKIVNGV MQYAPIGVFA LIAYVMAEQG VHVVGELAKV TAAVYVGLTL QILLVYFVLL KIYGIDPISF IKHAKDAMLT AFVTRSSEGT LPVTMRVAKE MGISEGIYSF TLPLGATINM DGTALYQGVC TFFIANALGS HLTVGQQLTI VLTAVLASIG TAGVPGAGAI MLAMVLHSVG LPLTDPNVAA AYAMILGIDA ILDMGRTMVN VTGNLTGTAI VAKTE

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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

BufferpH: 7.4
Component:
ConcentrationName
20.0 mMHEPES
250.0 mMsodium nitrate
0.8 mMDDM
1.0 mML-Asp
Sugar embeddingMaterial: ice
VitrificationCryogen name: ETHANE
DetailsSample was purified in the absence of substrate and the presence of sodium, and substrate was added just prior to freezing (~5s exposure).

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Average electron dose: 70.23 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.0 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 391453
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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