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- PDB-9dym: BEST1 + PABA intermediate state -

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Basic information

Entry
Database: PDB / ID: 9dym
TitleBEST1 + PABA intermediate state
ComponentsBestrophin-1
KeywordsMEMBRANE PROTEIN / calcium-activated chloride channel / para-aminobenzoic acid (PABA)-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
4-AMINOBENZOIC ACID / Bestrophin-1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.67 Å
AuthorsOwji, A.P. / Kittredge, A. / Zhang, Y. / Yang, T.
Funding support United States, 7items
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, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 8, 2025Provider: 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)339,48515
Polymers338,8025
Non-polymers68310
Water362
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-ID
11
22
33
44
55
66
77
88
99
1010
/ NCS ensembles :
ID
1
2
3
4
5
6
7
8
9
10

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Components

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


Mass: 67760.469 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
#3: Chemical ChemComp-PAB / 4-AMINOBENZOIC ACID


Mass: 137.136 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C7H7NO2 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationN

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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: BEST1 + PABA intermediate state / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.338 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 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: 1500 nm / Nominal defocus min: 800 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: 1634
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2PHENIX1.20.1_4487:model refinement
3Leginonimage acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 488010
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.67 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 40311 / 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
RefinementResolution: 2.67→2.67 Å / Cor.coef. Fo:Fc: 0.897 / SU B: 12.482 / SU ML: 0.222 / ESU R: 0.424
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.27443 --
obs0.27443 121529 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 57.808 Å2
Refinement stepCycle: 1 / Total: 15629
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0110.01216095
ELECTRON MICROSCOPYr_bond_other_d0.0010.01515123
ELECTRON MICROSCOPYr_angle_refined_deg1.3481.63921924
ELECTRON MICROSCOPYr_angle_other_deg1.5411.56734608
ELECTRON MICROSCOPYr_dihedral_angle_1_deg4.61851875
ELECTRON MICROSCOPYr_dihedral_angle_2_deg36.57121.591880
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.722152630
ELECTRON MICROSCOPYr_dihedral_angle_4_deg18.18915105
ELECTRON MICROSCOPYr_chiral_restr0.0770.21980
ELECTRON MICROSCOPYr_gen_planes_refined0.0080.0218042
ELECTRON MICROSCOPYr_gen_planes_other0.0020.024148
ELECTRON MICROSCOPYr_nbd_refined
ELECTRON MICROSCOPYr_nbd_other
ELECTRON MICROSCOPYr_nbtor_refined
ELECTRON MICROSCOPYr_nbtor_other
ELECTRON MICROSCOPYr_xyhbond_nbd_refined
ELECTRON MICROSCOPYr_xyhbond_nbd_other
ELECTRON MICROSCOPYr_metal_ion_refined
ELECTRON MICROSCOPYr_metal_ion_other
ELECTRON MICROSCOPYr_symmetry_vdw_refined
ELECTRON MICROSCOPYr_symmetry_vdw_other
ELECTRON MICROSCOPYr_symmetry_hbond_refined
ELECTRON MICROSCOPYr_symmetry_hbond_other
ELECTRON MICROSCOPYr_symmetry_metal_ion_refined
ELECTRON MICROSCOPYr_symmetry_metal_ion_other
ELECTRON MICROSCOPYr_mcbond_it6.3495.4347515
ELECTRON MICROSCOPYr_mcbond_other6.3475.4327514
ELECTRON MICROSCOPYr_mcangle_it9.348.2019385
ELECTRON MICROSCOPYr_mcangle_other9.348.2039386
ELECTRON MICROSCOPYr_scbond_it8.2536.588580
ELECTRON MICROSCOPYr_scbond_other8.2526.5818581
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other13.3129.46512540
ELECTRON MICROSCOPYr_long_range_B_refined18.31664772
ELECTRON MICROSCOPYr_long_range_B_other18.31664772
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumberRms dev position (Å)
11D243920.1
12B243920.1
21D241240.12
22E241240.12
31D241760.11
32A241760.11
41D241880.11
42C241880.11
51B241080.11
52E241080.11
61B240280.12
62A240280.12
71B240540.11
72C240540.11
81E242300.12
82A242300.12
91E243500.11
92C243500.11
101A242640.11
102C242640.11
LS refinement shellResolution: 3→3.078 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork0.778 8931 -
obs--100 %

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