[English] 日本語
Yorodumi
- EMDB-34167: CryoEM structure of cytosol-facing, substrate-bound ratGAT1 -

+
Open data


ID or keywords:

Loading...

-
Basic information

Entry
Database: EMDB / ID: EMD-34167
TitleCryoEM structure of cytosol-facing, substrate-bound ratGAT1
Map datarGAT1 EM map
Sample
  • Complex: Complex of epitope engineered ratGAT1-Fab9d5
    • Complex: ratGAT1
      • Protein or peptide: Sodium- and chloride-dependent GABA transporter 1
    • Complex: fragment antigen binding 9D5 heavy/light chain
      • Protein or peptide: Fragment antigen binding light chain
      • Protein or peptide: fragment antigen binding 9D5 heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHLORIDE ION
  • Ligand: SODIUM ION
  • Ligand: GAMMA-AMINO-BUTANOIC ACID
  • Ligand: CHOLESTEROL
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: water
KeywordsNeurotransmitter sodium symporter / GABA transporter / solute carrier 6 / secondary active transport / MEMBRANE PROTEIN
Function / homology
Function and homology information


Na+/Cl- dependent neurotransmitter transporters / Reuptake of GABA / neurotransmitter reuptake / gamma-aminobutyric acid:sodium:chloride symporter activity / sodium:chloride symporter activity / gamma-aminobutyric acid transmembrane transporter activity / gamma-aminobutyric acid import / inorganic anion import across plasma membrane / amino acid:sodium symporter activity / negative regulation of synaptic transmission, GABAergic ...Na+/Cl- dependent neurotransmitter transporters / Reuptake of GABA / neurotransmitter reuptake / gamma-aminobutyric acid:sodium:chloride symporter activity / sodium:chloride symporter activity / gamma-aminobutyric acid transmembrane transporter activity / gamma-aminobutyric acid import / inorganic anion import across plasma membrane / amino acid:sodium symporter activity / negative regulation of synaptic transmission, GABAergic / positive regulation of gamma-aminobutyric acid secretion / response to sucrose / response to purine-containing compound / sodium ion import across plasma membrane / associative learning / sodium ion transmembrane transport / : / GABA-ergic synapse / chloride transmembrane transport / response to organonitrogen compound / : / response to cocaine / learning / response to lead ion / synapse organization / response to organic cyclic compound / response to toxic substance / memory / response to calcium ion / presynapse / response to estradiol / presynaptic membrane / postsynaptic membrane / neuron projection / axon / neuronal cell body / cell surface / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Sodium:neurotransmitter symporter, GABA, GAT-1 / Sodium:neurotransmitter symporter family signature 2. / Sodium:neurotransmitter symporter family signature 1. / Sodium:neurotransmitter symporter / Sodium:neurotransmitter symporter superfamily / Sodium:neurotransmitter symporter family / Sodium:neurotransmitter symporter family profile.
Similarity search - Domain/homology
Sodium- and chloride-dependent GABA transporter 1
Similarity search - Component
Biological speciesRattus norvegicus (Norway rat) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsNayak SR / Joseph D / Penmatsa A
Funding support India, 1 items
OrganizationGrant numberCountry
Department of Biotechnology (DBT, India)IA/I/15/2/502063 India
CitationJournal: Nat Struct Mol Biol / Year: 2023
Title: Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism.
Authors: Smruti Ranjan Nayak / Deepthi Joseph / Georg Höfner / Archishman Dakua / Arunabh Athreya / Klaus T Wanner / Baruch I Kanner / Aravind Penmatsa /
Abstract: The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic ...The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.
History
DepositionAug 24, 2022-
Header (metadata) releaseMay 31, 2023-
Map releaseMay 31, 2023-
UpdateNov 15, 2023-
Current statusNov 15, 2023Processing site: PDBj / Status: Released

-
Structure visualization

Supplemental images

emd_34167.png

Downloads & links

-
Map

FileDownload / File: emd_34167.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationrGAT1 EM map
Voxel sizeX=Y=Z: 0.831 Å
Density
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-1.1288565 - 1.7588458
Average (Standard dev.)0.00005495065 (±0.029932575)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 299.16 Å
α=β=γ: 90.0 °

-
Supplemental data

-
Half map: half map B

Fileemd_34167_half_map_1.map
Annotationhalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Half map: half map A

Fileemd_34167_half_map_2.map
Annotationhalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

-
Sample components

+
Entire : Complex of epitope engineered ratGAT1-Fab9d5

EntireName: Complex of epitope engineered ratGAT1-Fab9d5
Components
  • Complex: Complex of epitope engineered ratGAT1-Fab9d5
    • Complex: ratGAT1
      • Protein or peptide: Sodium- and chloride-dependent GABA transporter 1
    • Complex: fragment antigen binding 9D5 heavy/light chain
      • Protein or peptide: Fragment antigen binding light chain
      • Protein or peptide: fragment antigen binding 9D5 heavy chain
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: CHLORIDE ION
  • Ligand: SODIUM ION
  • Ligand: GAMMA-AMINO-BUTANOIC ACID
  • Ligand: CHOLESTEROL
  • Ligand: PHOSPHATIDYLETHANOLAMINE
  • Ligand: water

+
Supramolecule #1: Complex of epitope engineered ratGAT1-Fab9d5

SupramoleculeName: Complex of epitope engineered ratGAT1-Fab9d5 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: rGAT1
Source (natural)Organism: Rattus norvegicus (Norway rat) / Location in cell: membrane
Molecular weightTheoretical: 111 kDa/nm

+
Supramolecule #2: ratGAT1

SupramoleculeName: ratGAT1 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

+
Supramolecule #3: fragment antigen binding 9D5 heavy/light chain

SupramoleculeName: fragment antigen binding 9D5 heavy/light chain / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3

+
Macromolecule #1: Sodium- and chloride-dependent GABA transporter 1

MacromoleculeName: Sodium- and chloride-dependent GABA transporter 1 / type: protein_or_peptide / ID: 1 / Details: ratGAT1 construct / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Rattus norvegicus (Norway rat)
Molecular weightTheoretical: 63.200676 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGDLPDRDTW KGRFDFLMSC VGYAIGLGNV WRFPYLCGKN GGGAFLIPYF LTLIFAGVPL FLLECSLGQY TSIGGLGVWK LAPMFKGVG LAAAVLSFWL NIYYIVIISW AIYYLYNSFT TTLPWKQCDN PWNTDRCFSN YSLVNTTNMT SAVVEFWERN M HQMTDGLD ...String:
MGDLPDRDTW KGRFDFLMSC VGYAIGLGNV WRFPYLCGKN GGGAFLIPYF LTLIFAGVPL FLLECSLGQY TSIGGLGVWK LAPMFKGVG LAAAVLSFWL NIYYIVIISW AIYYLYNSFT TTLPWKQCDN PWNTDRCFSN YSLVNTTNMT SAVVEFWERN M HQMTDGLD KPGQIRWPLA ITLAIAWVLV YFCIWKGVGW TGKVVYFSAT YPYIMLIILF FRGVTLPGAK EGILFYITPN FR KLSDSEV WLDAATQIFF SYGLGLGSLI ALGSYNSYHN NVYRDSIIVC CINSCTSMFA GFVIFSIVGF MAHVTKRSIA DVA ASGPGL AFLAYPEAVT QLPISPLWAI LFFSMLLMLG IDSQFCTVEG FITALVDEYP RLLRNRRELF IAAVCIVSYL IGLS NITQG GIYVFKLFDY YSASGMSLLF LVFFECVSIS WFYGVNRFSE DIRDMIGFPP CIWWKLCWSF FTPIIVAGVF LFSAV QMTP LTMGSYVFPK WGQGVGWLMA LSSMVLIPGY MAYMFLTLKG SLKQRLQVMI QPSEDIVRPE NGPEQPQAGS SASKEA YI

UniProtKB: Sodium- and chloride-dependent GABA transporter 1

+
Macromolecule #2: Fragment antigen binding light chain

MacromoleculeName: Fragment antigen binding light chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.306586 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: ENVLTQSPAI MSTSPGEKVT MTCRASSSVG SSYLHWYQQK SGASPKLWIY STSNLASGVP ARFSGSGSGT SYSLTISSVE AEDAATYYC QQFSGYPLTF GSGTKLEMKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD ...String:
ENVLTQSPAI MSTSPGEKVT MTCRASSSVG SSYLHWYQQK SGASPKLWIY STSNLASGVP ARFSGSGSGT SYSLTISSVE AEDAATYYC QQFSGYPLTF GSGTKLEMKR ADAAPTVSIF PPSSEQLTSG GASVVCFLNN FYPKDINVKW KIDGSERQNG V LNSWTDQD SKDSTYSMSS TLTLTKDEYE RHNSYTCEAT HKTSTSPIVK SFNRNE

+
Macromolecule #3: fragment antigen binding 9D5 heavy chain

MacromoleculeName: fragment antigen binding 9D5 heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.61943 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: EVQLVESGGG LVKPGGSLKL SCAASGFTFS SYAMSWVRQS PEKRLEWVAE ISSGGRYIYY SDTVTGRFTI SRDNARNILH LEMSSLRSE DTAMYYCARG EVRQRGFDYW GQGTTLTVSS AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG ...String:
EVQLVESGGG LVKPGGSLKL SCAASGFTFS SYAMSWVRQS PEKRLEWVAE ISSGGRYIYY SDTVTGRFTI SRDNARNILH LEMSSLRSE DTAMYYCARG EVRQRGFDYW GQGTTLTVSS AKTTAPSVYP LAPVCGDTTG SSVTLGCLVK GYFPEPVTLT W NSGSLSSG VHTFPAVLQS DLYTLSSSVT VTSSTWPSQS ITCNVAHPAS STKVDKKIEP R

+
Macromolecule #4: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 4 / Number of copies: 2 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose

+
Macromolecule #5: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

+
Macromolecule #6: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 6 / Number of copies: 1
Molecular weightTheoretical: 22.99 Da

+
Macromolecule #7: GAMMA-AMINO-BUTANOIC ACID

MacromoleculeName: GAMMA-AMINO-BUTANOIC ACID / type: ligand / ID: 7 / Number of copies: 1 / Formula: ABU
Molecular weightTheoretical: 103.12 Da
Chemical component information

ChemComp-ABU:
GAMMA-AMINO-BUTANOIC ACID

+
Macromolecule #8: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 8 / Number of copies: 6 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

+
Macromolecule #9: PHOSPHATIDYLETHANOLAMINE

MacromoleculeName: PHOSPHATIDYLETHANOLAMINE / type: ligand / ID: 9 / Number of copies: 1 / Formula: PTY
Molecular weightTheoretical: 734.039 Da
Chemical component information

ChemComp-PTY:
PHOSPHATIDYLETHANOLAMINE / phospholipid*YM

+
Macromolecule #10: water

MacromoleculeName: water / type: ligand / ID: 10 / Number of copies: 6 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

-
Experimental details

-
Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

-
Sample preparation

Concentration3.5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
20.0 mMTris
300.0 mMsodium chlorideNaCl
2.0 %Glycerol
1.0 mMDDM
0.1 mMCHS
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.76 kPa
VitrificationCryogen name: ETHANE
DetailsThe sample was a one to one complex of ratGAT1 with an antibody fragment and was homogenous.

-
Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 100.0 K / Max: 100.0 K
Specialist opticsPhase plate: OTHER / Spherical aberration corrector: Not applicable / Chromatic aberration corrector: No applicable / Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 18152 / Average exposure time: 2.7 sec. / Average electron dose: 50.09 e/Å2
Details: Images were collected in movie mode at 50 frames per image
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 105000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.0 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

+
Image processing

Particle selectionNumber selected: 5838634
Startup modelType of model: OTHER / Details: Alphafold2 model that was rebuilt into map.
Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3) / Software - details: Non-uniform refinement / Number images used: 872611
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final 3D classificationNumber classes: 5 / Avg.num./class: 200000 / Software - Name: cryoSPARC (ver. 3) / Software - details: Abinitio reconstruction
FSC plot (resolution estimation)

-
Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER / Overall B value: 165 / Target criteria: Correlation coefficient
Output model

PDB-8gnk:
CryoEM structure of cytosol-facing, substrate-bound ratGAT1

+
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://pdbjlvh1.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