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- EMDB-9321: BEST1 in a calcium-bound inactivated state -

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Basic information

Entry
Database: EMDB / ID: 9321
TitleBEST1 in a calcium-bound inactivated state
Map dataBEST1 in a calcium-bound inactivated state
SampleBEST1 (amino acids 2-405) in complex with calcium; inactivated conformation:
Bestrophin homolog / ligand
Function / homologyBestrophin / Bestrophin/UPF0187 / Bestrophin-1 / Bestrophin, RFP-TM, chloride channel / Stimuli-sensing channels / transepithelial chloride transport / detection of light stimulus involved in visual perception / chloride channel activity / chloride channel complex / regulation of calcium ion transport ...Bestrophin / Bestrophin/UPF0187 / Bestrophin-1 / Bestrophin, RFP-TM, chloride channel / Stimuli-sensing channels / 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 homolog
Function and homology information
SourceGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / 3.1 Å resolution
AuthorsMiller AN / Vaisey G / Long SB
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 ReportPDB-ID: 6n23

SummaryFull reportAbout validation report
DateDeposition: Nov 12, 2018 / Header (metadata) release: Jan 16, 2019 / Map release: Jan 23, 2019 / Last update: Jan 23, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6n23
  • Surface level: 0.018
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Supplemental images

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Map

Fileemd_9321.map.gz (map file in CCP4 format, 13501 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
150 pix
1.09 Å/pix.
= 163.2 Å
150 pix
1.09 Å/pix.
= 163.2 Å
150 pix
1.09 Å/pix.
= 163.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.088 Å
Density
Contour Level:0.018 (by author), 0.018 (movie #1):
Minimum - Maximum-0.020724207 - 0.047429092
Average (Standard dev.)0.0004417214 (0.0036553405)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions150150150
Origin0.00.00.0
Limit149.0149.0149.0
Spacing150150150
CellA=B=C: 163.20001 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0881.0881.088
M x/y/z150150150
origin x/y/z0.0000.0000.000
length x/y/z163.200163.200163.200
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0210.0470.000

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Supplemental data

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Sample components

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Entire BEST1 (amino acids 2-405) in complex with calcium; inactivated co...

EntireName: BEST1 (amino acids 2-405) in complex with calcium; inactivated conformation
Number of components: 3

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Component #1: protein, BEST1 (amino acids 2-405) in complex with calcium; inact...

ProteinName: BEST1 (amino acids 2-405) in complex with calcium; inactivated conformation
Recombinant expression: No
SourceSpecies: Gallus gallus (chicken)
Source (engineered)Expression System: Komagataella pastoris (fungus)

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Component #2: protein, Bestrophin homolog

ProteinName: Bestrophin homolog / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 47.614344 kDa
SourceSpecies: Gallus gallus (chicken)
Source (engineered)Expression System: Komagataella pastoris (fungus)

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Component #3: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 5 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 5 mg/ml / pH: 7.5
Support filmunspecified
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 293 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 76 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C5 (5 fold cyclic) / Number of projections: 294146
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 4RDQ
Chain ID: A

Overall bvalue: 87
Output model

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