[English] 日本語
Yorodumi
- EMDB-46587: EAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate buffer at OFS. -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-46587
TitleEAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate buffer at OFS.
Map dataEAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate at OFS
Sample
  • Complex: EAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate at OFS
    • Protein or peptide: human excitatory amino acid transporter 3
KeywordsEAAT3 / OFS / Sodium / TRANSPORT PROTEIN
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.07 Å
AuthorsQiu B / 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: bioRxiv / Year: 2024
Title: Structural basis of the excitatory amino acid transporter 3 substrate recognition.
Authors: Biao Qiu / Olga Boudker /
Abstract: Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only ...Excitatory amino acid transporters (EAATs) reside on cell surfaces and uptake substrates, including L-glutamate, L-aspartate, and D-aspartate, using ion gradients. Among five EAATs, EAAT3 is the only isoform that can efficiently transport L-cysteine, a substrate for glutathione synthesis. Recent work suggests that EAAT3 also transports the oncometabolite R-2-hydroxyglutarate (R-2HG). Here, we examined the structural basis of substrate promiscuity by determining the cryo-EM structures of EAAT3 bound to different substrates. We found that L-cysteine binds to EAAT3 in thiolate form, and EAAT3 recognizes different substrates by fine-tuning local conformations of the coordinating residues. However, using purified human EAAT3, we could not observe R-2HG binding or transport. Imaging of EAAT3 bound to L-cysteine revealed several conformational states, including an outward-facing state with a semi-open gate and a disrupted sodium-binding site. These structures illustrate that the full gate closure, coupled with the binding of the last sodium ion, occurs after substrate binding. Furthermore, we observed that different substrates affect how the transporter distributes between a fully outward-facing conformation and intermediate occluded states on a path to the inward-facing conformation, suggesting that translocation rates are substrate-dependent.
History
DepositionAug 14, 2024-
Header (metadata) releaseJan 22, 2025-
Map releaseJan 22, 2025-
UpdateJan 22, 2025-
Current statusJan 22, 2025Processing site: RCSB / Status: Released

-
Structure visualization

Supplemental images

emd_46587.png

Downloads & links

-
Map

FileDownload / File: emd_46587.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationEAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate at OFS
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.84 Å/pix.
x 320 pix.
= 270.176 Å		0.84 Å/pix.
x 320 pix.
= 270.176 Å		0.84 Å/pix.
x 320 pix.
= 270.176 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8443 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.34
Minimum - Maximum-2.2203834 - 3.1372082
Average (Standard dev.)0.0038262985 (±0.055259436)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 270.176 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Mask #1

Fileemd_46587_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: half map1

Fileemd_46587_half_map_1.map
Annotationhalf map1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: half map2

Fileemd_46587_half_map_2.map
Annotationhalf map2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

-
Entire : EAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate at OFS

EntireName: EAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate at OFS
Components
  • Complex: EAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate at OFS
    • Protein or peptide: human excitatory amino acid transporter 3

-
Supramolecule #1: EAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate at OFS

SupramoleculeName: EAAT3 in 200 mM NaCl and 10 mM R-2-hydroxyglutarate at OFS
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

-
Macromolecule #1: human excitatory amino acid transporter 3

MacromoleculeName: human excitatory amino acid transporter 3 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPMGKPARKG AEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PL IISSMIT GVAALDSNVS GKIGLRAVVY YFATTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVD AMLDLI RNMFPENLVQ AAFQQYKTKR ...String:
GPMGKPARKG AEWKRFLKNN WVLLSTVAAV VLGITTGVLV REHSNLSTLE KFYFAFPGEI LMRMLKLIIL PL IISSMIT GVAALDSNVS GKIGLRAVVY YFATTLIAVI LGIVLVVSIK PGVTQKVGEI ARTGSTPEVS TVD AMLDLI RNMFPENLVQ AAFQQYKTKR EEVKPPSDPE MTMTEESFTA VMTTAISKTK TKEYKIVGMY SDGI NVLGL IVFALVFGLV IGKMGEKGQI LVDFFNALSD ATMKIVQIIM WYMPLGILFL IAGCIIEVED WEIFR KLGL YMATVLTGLA IHSIVILPLI YFIVVRKNPF RFAMGMAQAL LTALMISSSS ATLPVTFRCA EENNQV DKR ITRFVLPVGA TINMDGTALY EAVAAVFIAQ LNDLDLGIGQ IITISITATS ASIGAAGVPQ AGLVTMV IV LSAVGLPAED VTLIIAVDCL LDRFRTMVNV LGDAFGTGIV EKLSKKELEQ MDVSSEVNIV NPFALEST I LDNEDSDTKK SYVNGGFAVD KSDTISFTQT SQF

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

BufferpH: 7.4
Details: 20 mM Hepes-Tris pH 7.4, 200 mM NaCl and 0.01% GDN, 10 mM R-2-hydroxyglutarate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE

-
Electron microscopy

MicroscopeFEI/PHILIPS CM300FEG/T
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.54 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.2 µm / Nominal defocus min: 0.8 µm

+
Image processing

Particle selectionNumber selected: 36362598
Startup modelType of model: OTHER / Details: Ab-initio
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.07 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4) / Number images used: 773970
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 4)
FSC plot (resolution estimation)

+
About Yorodumi

-
News

-
Feb 9, 2022. New format data for meta-information of EMDB entries

New format data for meta-information of EMDB entries

  • Version 3 of the EMDB header file is now the official format.
  • The previous official version 1.9 will be removed from the archive.

Related info.:EMDB header

External links:wwPDB to switch to version 3 of the EMDB data model

-
Aug 12, 2020. Covid-19 info

Covid-19 info

URL: https://pdbj.org/emnavi/covid19.php

New page: Covid-19 featured information page in EM Navigator.

Related info.:Covid-19 info / Mar 5, 2020. Novel coronavirus structure data

+
Mar 5, 2020. Novel coronavirus structure data

Novel coronavirus structure data

Related info.:Yorodumi Speices / Aug 12, 2020. Covid-19 info

External links:COVID-19 featured content - PDBj / Molecule of the Month (242)：Coronavirus Proteases

+
Jan 31, 2019. EMDB accession codes are about to change! (news from PDBe EMDB page)

EMDB accession codes are about to change! (news from PDBe EMDB page)

  • The allocation of 4 digits for EMDB accession codes will soon come to an end. Whilst these codes will remain in use, new EMDB accession codes will include an additional digit and will expand incrementally as the available range of codes is exhausted. The current 4-digit format prefixed with “EMD-” (i.e. EMD-XXXX) will advance to a 5-digit format (i.e. EMD-XXXXX), and so on. It is currently estimated that the 4-digit codes will be depleted around Spring 2019, at which point the 5-digit format will come into force.
  • The EM Navigator/Yorodumi systems omit the EMD- prefix.

Related info.:Q: What is EMD? / ID/Accession-code notation in Yorodumi/EM Navigator

External links:EMDB Accession Codes are Changing Soon! / Contact to PDBj

+
Jul 12, 2017. Major update of PDB

Major update of PDB

  • wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary.
  • This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated.
  • In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software).
  • Now, EM Navigator and Yorodumi are based on the updated data.

External links:wwPDB Remediation / Enriched Model Files Conforming to OneDep Data Standards Now Available in the PDB FTP Archive

-
Yorodumi

Thousand views of thousand structures

  • Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
  • This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
  • The word "yorodu" (or yorozu) is an old Japanese word meaning "ten thousand". "mi" (miru) is to see.

Related info.:EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Aug 31, 2016. New EM Navigator & Yorodumi / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi

Read more