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- EMDB-9323: BEST1 open state W287F mutant, calcium-bound -

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Basic information

Entry
Database: EMDB / ID: EMD-9323
TitleBEST1 open state W287F mutant, calcium-bound
Map dataBEST1 open state W287F mutant, calcium-bound
Sample
  • Complex: BEST1 open state W287F mutant, calcium-bound
    • Protein or peptide: Bestrophin homolog
  • Ligand: CALCIUM ION
Keywordsion channel / calcium activated chloride channel / eukaryotic membrane protein / anion channel / transport protein / ligand gated ion channel / MEMBRANE PROTEIN
Function / homologyBestrophin-1 / Stimuli-sensing channels / Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel / chloride channel activity / metal ion binding / membrane / Bestrophin 1
Function and homology information
Biological speciesGallus gallus (chicken)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.7 Å
AuthorsMiller AN / Vaisey G
Funding support United States, 2 items
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.
History
DepositionNov 12, 2018-
Header (metadata) releaseJan 23, 2019-
Map releaseJan 23, 2019-
UpdateNov 20, 2024-
Current statusNov 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6n25
  • Surface level: 0.029
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_9323.png

Downloads & links

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Map

FileDownload / File: emd_9323.map.gz / Format: CCP4 / Size: 12.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationBEST1 open state W287F mutant, calcium-bound
Projections & slices

Image control

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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.029 / Movie #1: 0.029
Minimum - Maximum-0.04458252 - 0.087014906
Average (Standard dev.)0.00034151186 (±0.0054769698)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions150150150
Spacing150150150
CellA=B=C: 163.20001 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS150150150
D min/max/mean-0.0450.0870.000

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Supplemental data

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Sample components

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Entire : BEST1 open state W287F mutant, calcium-bound

EntireName: BEST1 open state W287F mutant, calcium-bound
Components
  • Complex: BEST1 open state W287F mutant, calcium-bound
    • Protein or peptide: Bestrophin homolog
  • Ligand: CALCIUM ION

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Supramolecule #1: BEST1 open state W287F mutant, calcium-bound

SupramoleculeName: BEST1 open state W287F mutant, calcium-bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Gallus gallus (chicken)

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Macromolecule #1: Bestrophin homolog

MacromoleculeName: Bestrophin homolog / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Gallus gallus (chicken)
Molecular weightTheoretical: 40.722688 KDa
Recombinant expressionOrganism: Komagataella pastoris (fungus)
SequenceString: TVTYTNRVAD ARLGTFSQLL LQWKGSIYKL LYSEFLIFIS LYFAISLVYR LILSESQRLM FEKLALYCNS YAELIPVSFV LGFYVSLVV SRWWAQYESI PWPDRIMNLV SCNVDGEDEY GRLLRRTLMR YSNLCSVLIL RSVSTAVYKR FPSMEHVVRA G LMTPEEHK ...String:
TVTYTNRVAD ARLGTFSQLL LQWKGSIYKL LYSEFLIFIS LYFAISLVYR LILSESQRLM FEKLALYCNS YAELIPVSFV LGFYVSLVV SRWWAQYESI PWPDRIMNLV SCNVDGEDEY GRLLRRTLMR YSNLCSVLIL RSVSTAVYKR FPSMEHVVRA G LMTPEEHK KFESLNSPHN KFWIPCVWFS NLAVKARNEG RIRDSVLLQG ILNELNTLRS QCGRLYGYDW ISIPLVYTQV VT VAVYSFF LACLIGRQFL DPEKAYPGHE LDLFVPVFTF LQFFFYAGFL KVAEQLINPF GEDDDDFETN WLIDRNLQVS LMA VDEMHQ DLPILEKDLY WNEPDPQEGE EF

UniProtKB: Bestrophin 1

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 5 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 7.5
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Average exposure time: 10.0 sec. / Average electron dose: 76.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 265585
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 1 / Software - Name: RELION (ver. 2.1)

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Atomic model buiding 1

Initial modelPDB ID:

4rdq


Chain - Chain ID: A / Chain - Residue range: 2-367 / Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 55.9
Output model

PDB-6n25:
BEST1 open state W287F mutant, calcium-bound

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