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- EMDB-21433: bestrophin-2 (BEST2) calcium-unbound state 2 (EGTA only) -

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Basic information

Entry
Database: EMDB / ID: EMD-21433
Titlebestrophin-2 (BEST2) calcium-unbound state 2 (EGTA only)
Map databestrophin-2 (BEST2) calcium-free state 2 (EGTA only)
Sample
  • Cell: bestrophin-2 (BEST2) calcium-free state 2 (EGTA only)
    • Protein or peptide: Bestrophin
  • Ligand: CHLORIDE ION
  • Ligand: water
KeywordsChloride channel / MEMBRANE PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.33 Å
AuthorsOwji AP / Zhao Q
Funding support United States, 4 items
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, 2020-
Header (metadata) releaseMar 11, 2020-
Map releaseApr 8, 2020-
UpdateMar 6, 2024-
Current statusMar 6, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1
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  • Surface view colored by cylindrical radius
  • Surface level: 1
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  • Atomic models: PDB-6vx8
  • Surface level: 1
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6vx8
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Structure viewerEM map:
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Supplemental images

emd_21433.png

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Map

FileDownload / File: emd_21433.map.gz / Format: CCP4 / Size: 178 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationbestrophin-2 (BEST2) calcium-free state 2 (EGTA only)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.36 Å/pix.
x 360 pix.
= 128.761 Å		0.36 Å/pix.
x 360 pix.
= 128.761 Å		0.36 Å/pix.
x 360 pix.
= 128.761 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.35767 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 1.0 / Movie #1: 1
Minimum - Maximum-7.449581 - 11.186474
Average (Standard dev.)0.041554864 (±0.5557968)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions360360360
Spacing360360360
CellA=B=C: 128.7612 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.357669444444440.357669444444440.35766944444444
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z128.761128.761128.761
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ360360360
MAP C/R/S213
start NC/NR/NS000
NC/NR/NS360360360
D min/max/mean-7.45011.1860.042

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Supplemental data

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Additional map: Additional map

Fileemd_21433_additional.map
AnnotationAdditional map
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AxesZYX

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Histogram

Histogram (log scale)

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Half map: half-volume 1

Fileemd_21433_half_map_1.map
Annotationhalf-volume 1
Projections & Slices
AxesZYX

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Density Histograms

Histogram

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Half map: em-half-volume 2

Fileemd_21433_half_map_2.map
Annotationem-half-volume 2
Projections & Slices
AxesZYX

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Histogram (log scale)

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Sample components

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Entire : bestrophin-2 (BEST2) calcium-free state 2 (EGTA only)

EntireName: bestrophin-2 (BEST2) calcium-free state 2 (EGTA only)
Components
  • Cell: bestrophin-2 (BEST2) calcium-free state 2 (EGTA only)
    • Protein or peptide: Bestrophin
  • Ligand: CHLORIDE ION
  • Ligand: water

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Supramolecule #1: bestrophin-2 (BEST2) calcium-free state 2 (EGTA only)

SupramoleculeName: bestrophin-2 (BEST2) calcium-free state 2 (EGTA only) / type: cell / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (cattle)

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Macromolecule #1: Bestrophin

MacromoleculeName: Bestrophin / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Bos taurus (cattle)
Molecular weightTheoretical: 47.424754 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTVTYTARVA KARFGGFSKL LLLWRGSIYK LLWRELLCFL GLFMALSAAY RFVLTEEQKR YFEKLVLYCD RYASLIPVSF VLGFYVTLV VHRWWNQYLS MPLTDALMCV VVGTVHGHDE RGRLYRRTLM RYAGLSGVLI LRSVSTAVFK RFPTIDHVVE A GFMTREER ...String:
MTVTYTARVA KARFGGFSKL LLLWRGSIYK LLWRELLCFL GLFMALSAAY RFVLTEEQKR YFEKLVLYCD RYASLIPVSF VLGFYVTLV VHRWWNQYLS MPLTDALMCV VVGTVHGHDE RGRLYRRTLM RYAGLSGVLI LRSVSTAVFK RFPTIDHVVE A GFMTREER KKFENLNSSY NKYWVPCVWF CNLAAQARRE GRIRDNGAFK LLLEELNVFR SKCGMLFHYD WISVPLVYTQ VV TIAVYSY FLACLIGRQF LDPAQGYKDH DLDLCVPIFT LLQFFFYAGW LKVAEQLINP FGEDDDDFET NFLIDRCFQV SML AVDEMY DDLAMLEKDL YWDAAEARAP YTAATAFLMQ QPSFQGSTFD ITLAKEDMQF QRQDGLEAPL NEAHGDFLQR LLPV GTGMG TGGLL

UniProtKB: Bestrophin-2

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Macromolecule #2: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 2 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #3: water

MacromoleculeName: water / type: ligand / ID: 3 / Number of copies: 370 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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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

Concentration0.8 mg/mL
BufferpH: 7.8
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 69.67 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.33 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.13.2) / Number images used: 120642
Initial angle assignmentType: ANGULAR RECONSTITUTION
Final angle assignmentType: ANGULAR RECONSTITUTION

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