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- EMDB-47307: BEST2 + GABA closed state -

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Basic information

Entry
Database: EMDB / ID: EMD-47307
TitleBEST2 + GABA closed state
Map datasharpmap
Sample
  • Complex: BEST2 + GABA closed state
    • Protein or peptide: Bestrophin-2
  • Ligand: CALCIUM ION
  • Ligand: CHLORIDE ION
Keywordscalcium-activated chloride channel / GABA-bound anion channel / channel-activator complex / membrane protein
Function / homology
Function and homology information


intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / bicarbonate channel activity / chloride channel activity / ligand-gated monoatomic cation channel activity / membrane depolarization / chloride channel complex / Stimuli-sensing channels / sensory perception of smell / basolateral plasma membrane ...intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / bicarbonate channel activity / chloride channel activity / ligand-gated monoatomic cation channel activity / membrane depolarization / chloride channel complex / Stimuli-sensing channels / sensory perception of smell / basolateral plasma membrane / cilium / metal ion binding / plasma membrane
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: 2.27 Å
AuthorsOwji AP / Kittredge A / Zhang Y / Yang T
Funding support United States, 7 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 privateIrma T. Hirschl Trust CU20-4313 United States
Other privateResearch to Prevent Blindness (RPB) CU22-1892 United States
Simons FoundationSF349247 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129539 United States
CitationJournal: Nat Commun / Year: 2024
Title: Neurotransmitter-bound bestrophin channel structures reveal small molecule drug targeting sites for disease treatment.
Authors: Aaron P Owji / Jingyun Dong / Alec Kittredge / Jiali Wang / Yu Zhang / Tingting Yang /
Abstract: Best1 and Best2 are two members of the bestrophin family of anion channels critically involved in the prevention of retinal degeneration and maintenance of intraocular pressure, respectively. Here, ...Best1 and Best2 are two members of the bestrophin family of anion channels critically involved in the prevention of retinal degeneration and maintenance of intraocular pressure, respectively. Here, we solved glutamate- and γ-aminobutyric acid (GABA)-bound Best2 structures, which delineate an intracellular glutamate binding site and an extracellular GABA binding site on Best2, respectively, identified extracellular GABA as a permeable activator of Best2, and elucidated the co-regulation of Best2 by glutamate, GABA and glutamine synthetase in vivo. We further identified multiple small molecules as activators of the bestrophin channels. Extensive analyses were carried out for a potent activator, 4-aminobenzoic acid (PABA): PABA-bound Best1 and Best2 structures are solved and illustrate the same binding site as in GABA-bound Best2; PABA treatment rescues the functional deficiency of patient-derived Best1 mutations. Together, our results demonstrate the mechanism and potential of multiple small molecule candidates as clinically applicable drugs for bestrophin-associated diseases/conditions.
History
DepositionOct 14, 2024-
Header (metadata) releaseJan 8, 2025-
Map releaseJan 8, 2025-
UpdateJan 8, 2025-
Current statusJan 8, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47307.png

Downloads & links

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Map

FileDownload / File: emd_47307.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpmap
Projections & slices

Image control

Size
Brightness
Contrast
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

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-2.2541041 - 4.5733466
Average (Standard dev.)0.0010881417 (±0.11459634)
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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Additional map: fullmap

Fileemd_47307_additional_1.map
Annotationfullmap
Projections & Slices
AxesZYX

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Half map: halfA

Fileemd_47307_half_map_1.map
AnnotationhalfA
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: halfB

Fileemd_47307_half_map_2.map
AnnotationhalfB
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Sample components

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Entire : BEST2 + GABA closed state

EntireName: BEST2 + GABA closed state
Components
  • Complex: BEST2 + GABA closed state
    • Protein or peptide: Bestrophin-2
  • Ligand: CALCIUM ION
  • Ligand: CHLORIDE ION

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Supramolecule #1: BEST2 + GABA closed state

SupramoleculeName: BEST2 + GABA closed state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 286 KDa

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Macromolecule #1: Bestrophin-2

MacromoleculeName: Bestrophin-2 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.796898 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTVTYTARVA NARFGGFSQL LLLWRGSIYK LLWRELLCFL GFYMALSAAY RFVLTEGQKR YFEKLVIYCD QYASLIPVSF VLGFYVTLV VNRWWSQYLC MPLPDALMCV VAGTVHGRDD RGRLYRRTLM RYAGLSAVLI LRSVSTAVFK RFPTIDHVVE A GFMTREER ...String:
MTVTYTARVA NARFGGFSQL LLLWRGSIYK LLWRELLCFL GFYMALSAAY RFVLTEGQKR YFEKLVIYCD QYASLIPVSF VLGFYVTLV VNRWWSQYLC MPLPDALMCV VAGTVHGRDD RGRLYRRTLM RYAGLSAVLI LRSVSTAVFK RFPTIDHVVE A GFMTREER KKFENLNSSY NKYWVPCVWF SNLAAQARRE GRIRDNSALK LLLEELNVFR GKCGMLFHYD WISVPLVYTQ VV TIALYSY FLACLIGRQF LDPAQGYKDH DLDLCVPIFT LLQFFFYAGW LKVAEQLINP FGEDDDDFET NFLIDRNFQV SML AVDEMY DDLAVLEKDL YWDAAEARAP YTAATVFQLR QPSFQGSTFD ITLAKEDMQF QRLDGLDGPM GEAPGDFLQR LLPA GAGMV A

UniProtKB: Bestrophin-2a

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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: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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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 / 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
SoftwareName: Leginon
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 1130 / 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: 1.5 µm / Nominal defocus min: 0.8 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: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 54947
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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Atomic model buiding 1

Initial modelPDB ID:

8d1e


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

PDB-9dyk:
BEST2 + GABA closed state

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