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- PDB-6n28: BEST1 calcium-bound open state -

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Basic information

Entry
Database: PDB / ID: 6n28
TitleBEST1 calcium-bound open state
ComponentsBestrophin homolog
KeywordsMEMBRANE PROTEIN / ion channel / calcium activated chloride channel / eukaryotic membrane protein / anion channel / transport protein / ligand gated ion channel
Function / homology
Function and homology information


transepithelial chloride transport / detection of light stimulus involved in visual perception / chloride channel activity / chloride channel complex / regulation of calcium ion transport / basolateral plasma membrane / identical protein binding / metal ion binding
Bestrophin / Bestrophin/UPF0187 / Bestrophin-1 / Bestrophin, RFP-TM, chloride channel
Bestrophin 1
Biological speciesGallus gallus (chicken)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsMiller, A.N. / Vaisey, G. / Long, S.B.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM110396 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30 CA008748 United States
CitationJournal: Elife / Year: 2019
Title: Molecular mechanisms of gating in the calcium-activated chloride channel bestrophin.
Authors: Alexandria N Miller / George Vaisey / Stephen B Long /
Abstract: Bestrophin (BEST1-4) ligand-gated chloride (Cl) channels are activated by calcium (Ca). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural ...Bestrophin (BEST1-4) ligand-gated chloride (Cl) channels are activated by calcium (Ca). Mutation of BEST1 causes retinal disease. Partly because bestrophin channels have no sequence or structural similarity to other ion channels, the molecular mechanisms underlying gating are unknown. Here, we present a series of cryo-electron microscopy structures of chicken BEST1, determined at 3.1 Å resolution or better, that represent the channel's principal gating states. Unlike other channels, opening of the pore is due to the repositioning of tethered pore-lining helices within a surrounding protein shell that dramatically widens a neck of the pore through a concertina of amino acid rearrangements. The neck serves as both the activation and the inactivation gate. Ca binding instigates opening of the neck through allosteric means whereas inactivation peptide binding induces closing. An aperture within the otherwise wide pore controls anion permeability. The studies define a new molecular paradigm for gating among ligand-gated ion channels.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 12, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 23, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)204,00910
Polymers203,8095
Non-polymers2005
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Bestrophin homolog


Mass: 40761.727 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Gallus gallus (chicken) / Gene: BEST1 / Production host: Komagataella pastoris (fungus) / References: UniProt: E1C3A0
#2: Chemical
ChemComp-CA / CALCIUM ION / Calcium


Mass: 40.078 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Ca

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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 calcium-bound open state / Type: COMPLEX / Entity ID: 1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Gallus gallus (chicken)
Source (recombinant)Organism: Komagataella pastoris (fungus)
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K

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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: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 76 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
1RELION2.1particle selection
4RELION2.1CTF correction
7Coot0.8.9.1model fitting
9PHENIX1.13model refinement
10RELION2.1initial Euler assignment
11RELION2.1final Euler assignment
12RELION2.1classification
13RELION2.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41484 / Algorithm: FOURIER SPACE / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingB value: 55.9 / Protocol: OTHER / Space: REAL
Atomic model buildingPDB-ID: 4RDQ

4rdq


Pdb chain-ID: A / Pdb chain residue range: 2-367

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