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- PDB-6vx8: bestrophin-2 Ca2+- unbound state 2 (EGTA only) -

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Basic information

Entry
Database: PDB / ID: 6vx8
Titlebestrophin-2 Ca2+- unbound state 2 (EGTA only)
ComponentsBestrophin
KeywordsMEMBRANE PROTEIN / Chloride channel
Function / homology
Function and homology information


intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / Stimuli-sensing channels / bicarbonate channel activity / bicarbonate transport / chloride channel activity / ligand-gated monoatomic cation channel activity / chloride channel complex / basolateral plasma membrane / plasma membrane
Similarity search - Function
Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel
Similarity search - Domain/homology
Biological speciesBos taurus (cattle)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.33 Å
AuthorsOwji, A.P. / Zhao, Q. / Ji, C. / Kittredge, A. / Hopiavuori, A. / Fu, Z. / Ward, N. / Clarke, O. / Shen, Y. / Zhang, Y. ...Owji, A.P. / Zhao, Q. / Ji, C. / Kittredge, A. / Hopiavuori, A. / Fu, Z. / Ward, N. / Clarke, O. / Shen, Y. / Zhang, Y. / Hendrickson, W.A. / Yang, T.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)F31EY030763 United States
National Institutes of Health/National Eye Institute (NIH/NEI)EY025290 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM127652 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM107462 United States
CitationJournal: Nat Struct Mol Biol / Year: 2020
Title: Structural and functional characterization of the bestrophin-2 anion channel.
Authors: Aaron P Owji / Qingqing Zhao / Changyi Ji / Alec Kittredge / Austin Hopiavuori / Ziao Fu / Nancy Ward / Oliver B Clarke / Yin Shen / Yu Zhang / Wayne A Hendrickson / Tingting Yang /
Abstract: The bestrophin family of calcium (Ca)-activated chloride (Cl) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca, comprises four members ...The bestrophin family of calcium (Ca)-activated chloride (Cl) channels, which mediate the influx and efflux of monovalent anions in response to the levels of intracellular Ca, comprises four members in mammals (bestrophin 1-4). Here we report cryo-EM structures of bovine bestrophin-2 (bBest2) bound and unbound by Ca at 2.4- and 2.2-Å resolution, respectively. The bBest2 structure highlights four previously underappreciated pore-lining residues specifically conserved in Best2 but not in Best1, illustrating the differences between these paralogs. Structure-inspired electrophysiological analysis reveals that, although the channel is sensitive to Ca, it has substantial Ca-independent activity for Cl, reflecting the opening at the cytoplasmic restriction of the ion conducting pathway even when Ca is absent. Moreover, the ion selectivity of bBest2 is controlled by multiple residues, including those involved in gating.
History
DepositionFeb 21, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 8, 2020Provider: repository / Type: Initial release
Revision 1.1Apr 22, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)237,1596
Polymers237,1245
Non-polymers351
Water6,666370
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Bestrophin


Mass: 47424.754 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Bos taurus (cattle) / Gene: BEST2 / Cell line (production host): HEK293S / Production host: Homo sapiens (human) / References: UniProt: E1BF86
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 370 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: bestrophin-2 (BEST2) calcium-free state 2 (EGTA only) / Type: CELL / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Bos taurus (cattle)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293S
Buffer solutionpH: 7.8
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 69.67 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
13cryoSPARC2.13.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 120642 / Symmetry type: POINT

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