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- EMDB-45916: Best1 + GABA intermediate state 1 -

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Basic information

Entry
Database: EMDB / ID: EMD-45916
TitleBest1 + GABA intermediate state 1
Map data
Sample
  • Complex: GABA-bound Homopentameric complex of Best1 anion channel in intermediate state 1 ICCCC
    • Protein or peptide: Bestrophin-1
  • Ligand: CALCIUM ION
  • Ligand: GAMMA-AMINO-BUTANOIC ACID
Keywordscalcium-activated chloride channel / GABA type A receptor / GABA-bound anion channel / channel-activator complex / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.42 Å
AuthorsOwji AP / Kittredge A / Zhang Y / Yang T
Funding support United States, 5 items
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, 2024-
Header (metadata) releaseSep 25, 2024-
Map releaseSep 25, 2024-
UpdateSep 25, 2024-
Current statusSep 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45916.png

Downloads & links

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Map

FileDownload / File: emd_45916.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 400 pix.
= 332. Å		0.83 Å/pix.
x 400 pix.
= 332. Å		0.83 Å/pix.
x 400 pix.
= 332. Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-1.7264929 - 4.1342278
Average (Standard dev.)-0.0002833003 (±0.09974449)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 332.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_45916_msk_1.map
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Additional map: full map

Fileemd_45916_additional_1.map
Annotationfull_map
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Half map: half B

Fileemd_45916_half_map_1.map
Annotationhalf_B
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Half map: half A

Fileemd_45916_half_map_2.map
Annotationhalf_A
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Sample components

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Entire : GABA-bound Homopentameric complex of Best1 anion channel in inter...

EntireName: GABA-bound Homopentameric complex of Best1 anion channel in intermediate state 1 ICCCC
Components
  • Complex: GABA-bound Homopentameric complex of Best1 anion channel in intermediate state 1 ICCCC
    • Protein or peptide: Bestrophin-1
  • Ligand: CALCIUM ION
  • Ligand: GAMMA-AMINO-BUTANOIC ACID

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Supramolecule #1: GABA-bound Homopentameric complex of Best1 anion channel in inter...

SupramoleculeName: GABA-bound Homopentameric complex of Best1 anion channel in intermediate state 1 ICCCC
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 / Details: GABA-activated structure
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 337.5 KDa

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Macromolecule #1: Bestrophin-1

MacromoleculeName: Bestrophin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 67.629273 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: TITYTSQVAN ARLGSFSRLL LCWRGSIYKL LYGEFLIFLL CYYIIRFIYR LALTEEQQLM FEKLTLYCDS YIQLIPISFV LGFYVTLVV TRWWNQYENL PWPDRLMSLV SGFVEGKDEQ GRLLRRTLIR YANLGNVLIL RSVSTAVYKR FPSAQHLVQA G FMTPAEHK ...String:
TITYTSQVAN ARLGSFSRLL LCWRGSIYKL LYGEFLIFLL CYYIIRFIYR LALTEEQQLM FEKLTLYCDS YIQLIPISFV LGFYVTLVV TRWWNQYENL PWPDRLMSLV SGFVEGKDEQ GRLLRRTLIR YANLGNVLIL RSVSTAVYKR FPSAQHLVQA G FMTPAEHK QLEKLSLPHN MFWVPWVWFA NLSMKAWLGG RIRDPILLQS LLNEMNTLRT QCGHLYAYDW ISIPLVYTQV VT VAVYSFF LTCLVGRQFL NPAKAYPGHE LDLVVPVFTF LQFFFYVGWL KVAEQLINPF GEDDDDFETN WIVDRNLQVS LLA VDEMHQ DLPRMEPDMY WNKPEPQPPY TAASAQFRRA SFMGSTFNIS LNKEEMEFQP NQEDEEDAHA GIIGRFLGLQ SHDH HPPRA NSRTKLLWPK RESLLHEGLP KNHKAAKQNV RGQEDNKAWK LKAVDAFKSA PLYQRPGYYS APQTPLSPTP MFFPL EPSA PSKLHSVTGI DTKDKSLKTV SSGAKKSFEL LSESDGALME HPEVSQVRRK TVEFNLTDMP EIPENHLKEP LEQSPT NIH TTLKDHMDPY WALENRDEAH S

UniProtKB: Bestrophin-1

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 5 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: GAMMA-AMINO-BUTANOIC ACID

MacromoleculeName: GAMMA-AMINO-BUTANOIC ACID / type: ligand / ID: 3 / Number of copies: 4 / Formula: ABU
Molecular weightTheoretical: 103.12 Da
Chemical component information

ChemComp-ABU:
GAMMA-AMINO-BUTANOIC 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

Concentration5 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
200.0 mMNaClsodium chloride
40.0 mMHEPES4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
0.008 % w/vGDNglyco-diosgenin
GridModel: UltrAuFoil R0./1 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 713 / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 105000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER / Details: ab initio reconstruction
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.42 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 173379
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelPDB ID:

8d1i


Chain - Chain ID: A / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementProtocol: RIGID BODY FIT
Output model

PDB-9ctr:
Best1 + GABA intermediate state 1

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