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- PDB-9dyh: BEST2 + glutamate open state -

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Basic information

Entry
Database: PDB / ID: 9dyh
TitleBEST2 + glutamate open state
ComponentsBestrophin-2a
KeywordsMEMBRANE PROTEIN / calcium-activated chloride channel / Glutamate-bound anion channel / channel-activator complex
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.15 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 15, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Bestrophin-2a
E: Bestrophin-2a
D: Bestrophin-2a
B: Bestrophin-2a
C: Bestrophin-2a
hetero molecules


Theoretical massNumber of molelcules
Total (without water)286,14410
Polymers285,9435
Non-polymers2005
Water00
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-2a / Vitelliform macular dystrophy 2-like protein 1


Mass: 57188.637 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BEST2, VMD2L1 / Cell line (production host): HEK293 / Production host: Homo sapiens (human) / References: UniProt: Q8NFU1
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN
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: BEST2 + glutamate open state / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.286 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: 2500 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Image recordingElectron dose: 58.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 6625
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
9PHENIX1.20.1_4487:model refinement
13Leginon3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3529672
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.15 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 315547 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 8D1G

8d1g


Pdb chain-ID: A / Accession code: 8D1G / Source name: PDB / Type: experimental model
RefinementResolution: 3.15→3.15 Å / Cor.coef. Fo:Fc: 0.897 / SU B: 26.593 / SU ML: 0.433 / ESU R: 1.139
Stereochemistry target values: MAXIMUM LIKELIHOOD WITH PHASES
Details: HYDROGENS HAVE BEEN ADDED IN THE RIDING POSITIONS
RfactorNum. reflection% reflection
Rwork0.36376 --
obs0.36376 73360 100 %
Solvent computationSolvent model: PARAMETERS FOR MASK CACLULATION
Displacement parametersBiso mean: 102.177 Å2
Refinement stepCycle: 1 / Total: 14541
Refine LS restraints
Refine-IDTypeDev idealDev ideal targetNumber
ELECTRON MICROSCOPYr_bond_refined_d0.0090.01214985
ELECTRON MICROSCOPYr_bond_other_d0.030.01514140
ELECTRON MICROSCOPYr_angle_refined_deg1.2091.63320345
ELECTRON MICROSCOPYr_angle_other_deg1.5071.57232295
ELECTRON MICROSCOPYr_dihedral_angle_1_deg5.08551740
ELECTRON MICROSCOPYr_dihedral_angle_2_deg32.33720.588850
ELECTRON MICROSCOPYr_dihedral_angle_3_deg16.623152410
ELECTRON MICROSCOPYr_dihedral_angle_4_deg18.20315120
ELECTRON MICROSCOPYr_chiral_restr0.0710.21850
ELECTRON MICROSCOPYr_gen_planes_refined0.0060.0216595
ELECTRON MICROSCOPYr_gen_planes_other0.0020.023945
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_it11.20210.0726960
ELECTRON MICROSCOPYr_mcbond_other11.20310.076959
ELECTRON MICROSCOPYr_mcangle_it17.24215.0488660
ELECTRON MICROSCOPYr_mcangle_other17.24115.058661
ELECTRON MICROSCOPYr_scbond_it12.77911.4828025
ELECTRON MICROSCOPYr_scbond_other12.77811.4838026
ELECTRON MICROSCOPYr_scangle_it
ELECTRON MICROSCOPYr_scangle_other20.50416.73911676
ELECTRON MICROSCOPYr_long_range_B_refined30.9762702
ELECTRON MICROSCOPYr_long_range_B_other30.9762703
ELECTRON MICROSCOPYr_rigid_bond_restr
ELECTRON MICROSCOPYr_sphericity_free
ELECTRON MICROSCOPYr_sphericity_bonded
Refine LS restraints NCS

Refine-ID: ELECTRON MICROSCOPY / Type: interatomic distance / Rms dev position: 0.02 Å / Weight position: 0.05

Ens-IDDom-IDAuth asym-IDNumber
11A24538
12B24538
21A24540
22E24540
31A24550
32C24550
41A24538
42D24538
51B24532
52E24532
61B24542
62C24542
71B24534
72D24534
81E24542
82C24542
91E24532
92D24532
101C24540
102D24540
LS refinement shellResolution: 3.2→3.283 Å / Total num. of bins used: 20
RfactorNum. reflection% reflection
Rfree0 0 -
Rwork1.197 5355 -
obs--100 %

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