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- EMDB-44528: Ancestral uncoupled aspartate transporter in complex with L-aspartate -

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Basic information

Entry
Database: EMDB / ID: EMD-44528
TitleAncestral uncoupled aspartate transporter in complex with L-aspartate
Map data
Sample
  • Complex: Ancestral uncoupled aspartate transporter in complex with L-aspartate
    • Protein or peptide: Aspartate transporter
  • Ligand: ASPARTIC ACID
KeywordsTransporter / ion-uncoupled / ancestral / TRANSPORT PROTEIN
Biological speciesPyrococcus horikoshii (archaea) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.4 Å
AuthorsReddy KD / Boudker O
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R37NS134865 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)F32NS102325 United States
CitationJournal: bioRxiv / Year: 2024
Title: Evolutionary analysis reveals the origin of sodium coupling in glutamate transporters.
Authors: Krishna D Reddy / Burha Rasool / Farideh Badichi Akher / Nemanja Kutlešić / Swati Pant / Olga Boudker /
Abstract: Secondary active membrane transporters harness the energy of ion gradients to concentrate their substrates. Homologous transporters evolved to couple transport to different ions in response to ...Secondary active membrane transporters harness the energy of ion gradients to concentrate their substrates. Homologous transporters evolved to couple transport to different ions in response to changing environments and needs. The bases of such diversification, and thus principles of ion coupling, are unexplored. Employing phylogenetics and ancestral protein reconstruction, we investigated sodium-coupled transport in prokaryotic glutamate transporters, a mechanism ubiquitous across life domains and critical to neurotransmitter recycling in humans. We found that the evolutionary transition from sodium-dependent to independent substrate binding to the transporter preceded changes in the coupling mechanism. Structural and functional experiments suggest that the transition entailed allosteric mutations, making sodium binding dispensable without affecting ion-binding sites. Allosteric tuning of transporters' energy landscapes might be a widespread route of their functional diversification.
History
DepositionApr 19, 2024-
Header (metadata) releaseMar 12, 2025-
Map releaseMar 12, 2025-
UpdateMar 12, 2025-
Current statusMar 12, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_44528.png

Downloads & links

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Map

FileDownload / File: emd_44528.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 360 pix.
= 299.88 Å		0.83 Å/pix.
x 360 pix.
= 299.88 Å		0.83 Å/pix.
x 360 pix.
= 299.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.833 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.166
Minimum - Maximum-0.0005135915 - 2.0982034
Average (Standard dev.)0.00059621985 (±0.019803712)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 299.88 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_44528_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_44528_half_map_2.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : Ancestral uncoupled aspartate transporter in complex with L-aspartate

EntireName: Ancestral uncoupled aspartate transporter in complex with L-aspartate
Components
  • Complex: Ancestral uncoupled aspartate transporter in complex with L-aspartate
    • Protein or peptide: Aspartate transporter
  • Ligand: ASPARTIC ACID

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Supramolecule #1: Ancestral uncoupled aspartate transporter in complex with L-aspartate

SupramoleculeName: Ancestral uncoupled aspartate transporter in complex with L-aspartate
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Pyrococcus horikoshii (archaea)

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Macromolecule #1: Aspartate transporter

MacromoleculeName: Aspartate transporter / type: protein_or_peptide / ID: 1
Details: C-terminal thrombin cleavage site, Twin-Strep tag, ALFA tag, and (HIS)10 tag. Engineered with loops and tails from NCBI KJS87745.1
Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 43.910488 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MSLYHKILIG FVLGVIVGLI FGDKAEFIKP LGDIFLRLLK MIVVPLVFST IVTGIASMGD VKKLGRIGAK TLIYYMITTT LAVTIGLIL ANIFKPGKGL SLGEIHEVAH PNAPSFTETL LNMIPTNPFE AMAEGNMLQI IVFAIFFGIA LALMGEKAEP V KKFFDSAS ...String:
MSLYHKILIG FVLGVIVGLI FGDKAEFIKP LGDIFLRLLK MIVVPLVFST IVTGIASMGD VKKLGRIGAK TLIYYMITTT LAVTIGLIL ANIFKPGKGL SLGEIHEVAH PNAPSFTETL LNMIPTNPFE AMAEGNMLQI IVFAIFFGIA LALMGEKAEP V KKFFDSAS EVMFKITDIV MKFAPYGVFA LMAWTVGKYG LDVLAPLGKL ILTVYLGCII HILIVYTLLL RFLCKINPLR FF KKIKEAM LVAFSTCSSA ATLPVTMRVA EELGVPESIA SFTLPLGATI NMDGTALYQG VAAIFVAQAY GVELTLGQQL TIV LTAVLA SIGTAGVPGA GLVMLTMVLT SVGLPLEGIA LIAGIDRILD MARTTVNVTG DLVATAIVAR TENELNREMT ATPL EVLES KTIAL

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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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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 20 mM HEPES, 100 mM NMDG-Cl, 1 mM L-Asp
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.9 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71731
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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