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- EMDB-27130: hBest2 Ca2+-unbound closed state -

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Basic information

Entry
Database: EMDB / ID: EMD-27130
TitlehBest2 Ca2+-unbound closed state
Map datadensity modified (phenix.resolve) and resampled in coot (resample factor = 2)
Sample
  • Complex: hBest2 Ca2+-unbound closed state
    • Protein or peptide: Bestrophin-2
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 2-[2-[(1~{S},2~{S},4~{S},5'~{R},6~{R},7~{S},8~{R},9~{S},12~{S},13~{R},16~{S})-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.0^{2,9}.0^{4,8}.0^{13,18}]icos-18-ene-6,2'-oxane]-16-yl]oxyethyl]propane-1,3-diol
  • Ligand: CHLORIDE IONChloride
  • Ligand: water
KeywordsIon channel / chloride channel / anion channel / pentamer / TRANSPORT PROTEIN
Function / homology
Function and homology information


chloride channel activity / membrane depolarization / chloride channel complex / cilium / Stimuli-sensing channels / sensory perception of smell / plasma membrane
Similarity search - Function
Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.94 Å
AuthorsOwji AP / Kittredge A / Hendrickson WA / Tingting Y
Funding support United States, 3 items
OrganizationGrant numberCountry
National Institutes of Health/National Eye Institute (NIH/NEI)EY030763-03 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM107462-08 United States
National Institutes of Health/National Eye Institute (NIH/NEI)GM127652-06 United States
CitationJournal: Nat Commun / Year: 2022
Title: Structures and gating mechanisms of human bestrophin anion channels.
Authors: Aaron P Owji / Jiali Wang / Alec Kittredge / Zada Clark / Yu Zhang / Wayne A Hendrickson / Tingting Yang /
Abstract: Bestrophin-1 (Best1) and bestrophin-2 (Best2) are two members of the bestrophin family of calcium (Ca)-activated chloride (Cl) channels with critical involvement in ocular physiology and direct ...Bestrophin-1 (Best1) and bestrophin-2 (Best2) are two members of the bestrophin family of calcium (Ca)-activated chloride (Cl) channels with critical involvement in ocular physiology and direct pathological relevance. Here, we report cryo-EM structures of wild-type human Best1 and Best2 in various states at up to 1.8 Å resolution. Ca-bound Best1 structures illustrate partially open conformations at the two Ca-dependent gates of the channels, in contrast to the fully open conformations observed in Ca-bound Best2, which is in accord with the significantly smaller currents conducted by Best1 in electrophysiological recordings. Comparison of the closed and open states reveals a C-terminal auto-inhibitory segment (AS), which constricts the channel concentrically by wrapping around the channel periphery in an inter-protomer manner and must be released to allow channel opening. Our results demonstrate that removing the AS from Best1 and Best2 results in truncation mutants with similar activities, while swapping the AS between Best1 and Best2 results in chimeric mutants with swapped activities, underlying a key role of the AS in determining paralog specificity among bestrophins.
History
DepositionMay 27, 2022-
Header (metadata) releaseJul 13, 2022-
Map releaseJul 13, 2022-
UpdateFeb 14, 2024-
Current statusFeb 14, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_27130.png

Downloads & links

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Map

FileDownload / File: emd_27130.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdensity modified (phenix.resolve) and resampled in coot (resample factor = 2)
Voxel sizeX=Y=Z: 0.39194 Å
Density
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-5.032693 - 9.774343500000001
Average (Standard dev.)-0.000000000529599 (±0.5881741)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 112.87872 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Difference map obtained by vop subtract from Ca2...

Fileemd_27130_additional_1.map
AnnotationDifference map obtained by vop subtract from Ca2 -bound map after filtering to 1.9A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: full map

Fileemd_27130_additional_2.map
Annotationfull map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: cryosparc half map 1

Fileemd_27130_half_map_1.map
Annotationcryosparc half map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: cryosparc half map 2

Fileemd_27130_half_map_2.map
Annotationcryosparc half map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : hBest2 Ca2+-unbound closed state

EntireName: hBest2 Ca2+-unbound closed state
Components
  • Complex: hBest2 Ca2+-unbound closed state
    • Protein or peptide: Bestrophin-2
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: 2-[2-[(1~{S},2~{S},4~{S},5'~{R},6~{R},7~{S},8~{R},9~{S},12~{S},13~{R},16~{S})-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.0^{2,9}.0^{4,8}.0^{13,18}]icos-18-ene-6,2'-oxane]-16-yl]oxyethyl]propane-1,3-diol
  • Ligand: CHLORIDE IONChloride
  • Ligand: water

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Supramolecule #1: hBest2 Ca2+-unbound closed state

SupramoleculeName: hBest2 Ca2+-unbound closed state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 234 KDa

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Macromolecule #1: Bestrophin-2

MacromoleculeName: Bestrophin-2 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 46.796898 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTVTYTARVA NARFGGFSQL LLLWRGSIYK LLWRELLCFL GFYMALSAAY RFVLTEGQKR YFEKLVIYCD QYASLIPVSF VLGFYVTLV VNRWWSQYLC MPLPDALMCV VAGTVHGRDD RGRLYRRTLM RYAGLSAVLI LRSVSTAVFK RFPTIDHVVE A GFMTREER ...String:
MTVTYTARVA NARFGGFSQL LLLWRGSIYK LLWRELLCFL GFYMALSAAY RFVLTEGQKR YFEKLVIYCD QYASLIPVSF VLGFYVTLV VNRWWSQYLC MPLPDALMCV VAGTVHGRDD RGRLYRRTLM RYAGLSAVLI LRSVSTAVFK RFPTIDHVVE A GFMTREER KKFENLNSSY NKYWVPCVWF SNLAAQARRE GRIRDNSALK LLLEELNVFR GKCGMLFHYD WISVPLVYTQ VV TIALYSY FLACLIGRQF LDPAQGYKDH DLDLCVPIFT LLQFFFYAGW LKVAEQLINP FGEDDDDFET NFLIDRNFQV SML AVDEMY DDLAVLEKDL YWDAAEARAP YTAATVFQLR QPSFQGSTFD ITLAKEDMQF QRLDGLDGPM GEAPGDFLQR LLPA GAGMV A

UniProtKB: Bestrophin-2

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Macromolecule #2: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 2 / Number of copies: 45 / Formula: MC3
Molecular weightTheoretical: 677.933 Da
Chemical component information

ChemComp-MC3:
1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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Macromolecule #3: 2-[2-[(1~{S},2~{S},4~{S},5'~{R},6~{R},7~{S},8~{R},9~{S},12~{S},13...

MacromoleculeName: 2-[2-[(1~{S},2~{S},4~{S},5'~{R},6~{R},7~{S},8~{R},9~{S},12~{S},13~{R},16~{S})-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.0^{2,9}.0^{4,8}.0^{13,18}]icos-18-ene-6,2'-oxane]-16-yl]oxyethyl]propane-1,3-diol
type: ligand / ID: 3 / Number of copies: 5 / Formula: DU0
Molecular weightTheoretical: 516.752 Da
Chemical component information

ChemComp-DU0:
2-[2-[(1~{S},2~{S},4~{S},5'~{R},6~{R},7~{S},8~{R},9~{S},12~{S},13~{R},16~{S})-5',7,9,13-tetramethylspiro[5-oxapentacyclo[10.8.0.0^{2,9}.0^{4,8}.0^{13,18}]icos-18-ene-6,2'-oxane]-16-yl]oxyethyl]propane-1,3-diol

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Macromolecule #4: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 605 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / 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

Concentration5 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
40.0 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
0.005 %glyco-diosgenin
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK IV
DetailshBest2 Ca2+-unbound closed state

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 3446 / Average electron dose: 58.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 2853381
Startup modelType of model: OTHER / Details: Ab initio
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 1.94 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 656860

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