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- PDB-9ctt: Best1 + GABA closed state -

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Basic information

Entry
Database: PDB / ID: 9ctt
TitleBest1 + GABA closed state
ComponentsBestrophin-1
KeywordsMEMBRANE PROTEIN / calcium-activated chloride channel / GABA type A receptor / GABA-bound anion channel / channel-activator complex
Function / homology
Function and homology information


membrane microdomain / bicarbonate channel activity / transepithelial chloride transport / gamma-aminobutyric acid secretion, neurotransmission / detection of light stimulus involved in visual perception / ligand-gated channel activity / intracellularly calcium-gated chloride channel activity / bicarbonate transmembrane transporter activity / glutamate secretion / chloride transport ...membrane microdomain / bicarbonate channel activity / transepithelial chloride transport / gamma-aminobutyric acid secretion, neurotransmission / detection of light stimulus involved in visual perception / ligand-gated channel activity / intracellularly calcium-gated chloride channel activity / bicarbonate transmembrane transporter activity / glutamate secretion / chloride transport / chloride channel activity / protein complex oligomerization / regulation of calcium ion transport / chloride channel complex / visual perception / basal plasma membrane / regulation of synaptic plasticity / Stimuli-sensing channels / presynapse / monoatomic ion transmembrane transport / basolateral plasma membrane / identical protein binding / membrane / plasma membrane / cytosol
Similarity search - Function
Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsOwji, A.P. / Kittredge, A. / Zhang, Y. / Yang, T.
Funding support United States, 5items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM149252 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM127652 United States
National Institutes of Health/National Eye Institute (NIH/NEI)R24EY028758 United States
Other privateCU20-4313 United States
Other privateCU22-1892 United States
CitationJournal: Nat Commun / Year: 2024
Title: GAD65 tunes the functions of Best1 as a GABA receptor and a neurotransmitter conducting channel.
Authors: Jiali Wang / Aaron P Owji / Alec Kittredge / Zada Clark / Yu Zhang / Tingting Yang /
Abstract: Bestrophin-1 (Best1) is an anion channel genetically linked to vision-threatening retinal degenerative channelopathies. Here, we identify interactions between Best1 and both isoforms of glutamic acid ...Bestrophin-1 (Best1) is an anion channel genetically linked to vision-threatening retinal degenerative channelopathies. Here, we identify interactions between Best1 and both isoforms of glutamic acid decarboxylases (GAD65 and GAD67), elucidate the distinctive influences of GAD65 and GAD67 on Best1's permeability to various anions/neurotransmitters, discover the functionality of Best1 as a γ-Aminobutyric acid (GABA) type A receptor, and solve the structure of GABA-bound Best1. GAD65 and GAD67 both promote Best1-mediated Cl currents, but only GAD65 drastically enhances the permeability of Best1 to glutamate and GABA, for which GAD67 has no effect. GABA binds to Best1 on an extracellular site and stimulates Best1-mediated Cl currents at the nano-molar concentration level. The physiological role of GAD65 as a cell type-specific binding partner and facilitator of Best1 is demonstrated in retinal pigment epithelial cells. Together, our results reveal critical regulators of Best1 and inform a network of membrane transport metabolons formed between bestrophin channels and glutamate metabolic enzymes.
History
DepositionJul 25, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 25, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
D: Bestrophin-1
B: Bestrophin-1
E: Bestrophin-1
A: Bestrophin-1
C: Bestrophin-1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)338,34710
Polymers338,1465
Non-polymers2005
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Bestrophin-1 / TU15B / Vitelliform macular dystrophy protein 2


Mass: 67629.273 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BEST1, VMD2 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: O76090
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: GABA-bound Homopentameric complex of Best1 anion channel in fully open fffff state
Type: COMPLEX / Details: GABA-activated structure / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.3375 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
240 mM4-(2-hydroxyethyl)-1-piperazineethanesulfonic acidHEPES1
30.008 % w/vglyco-diosgeninGDN1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: UltrAuFoil R0./1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 713
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM softwareName: PHENIX / Version: 1.20.1_4487: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 52529 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 8D1I

8d1i


Pdb chain-ID: A / Accession code: 8D1I / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00316065
ELECTRON MICROSCOPYf_angle_d0.51821885
ELECTRON MICROSCOPYf_dihedral_angle_d3.9522080
ELECTRON MICROSCOPYf_chiral_restr0.0382375
ELECTRON MICROSCOPYf_plane_restr0.0042750

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