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- EMDB-27136: bBest2_345 Ca2+-bound open state -

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Basic information

Entry
Database: EMDB / ID: EMD-27136
TitlebBest2_345 Ca2+-bound open state
Map datadensity modified (phenix.resolve) and resampled in coot (resample factor = 1.5)
Sample
  • Complex: bBest2_345 Ca2+-bound open state
    • Protein or peptide: Bestrophin
  • Ligand: CALCIUM ION
  • 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 ION
  • Ligand: water
KeywordsIon channel / chloride channel / anion channel / pentamer / TRANSPORT PROTEIN
Function / homology
Function and homology information


Stimuli-sensing channels / intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / bicarbonate channel activity / bicarbonate transport / ligand-gated monoatomic cation channel activity / chloride 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 (domestic cattle)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.93 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_27136.png

Downloads & links

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Map

FileDownload / File: emd_27136.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationdensity modified (phenix.resolve) and resampled in coot (resample factor = 1.5)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)X (Row.)Y (Col.)
0.55 Å/pix.
x 200 pix.
= 109.56 Å		0.55 Å/pix.
x 200 pix.
= 109.56 Å		0.55 Å/pix.
x 200 pix.
= 109.56 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.5478 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.6
Minimum - Maximum-4.7760363 - 17.246784000000002
Average (Standard dev.)0.000000000058392 (±0.61153597)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderYXZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 109.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: full map

Fileemd_27136_additional_1.map
Annotationfull map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_27136_half_map_1.map
Annotationhalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1

Fileemd_27136_half_map_2.map
Annotationhalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : bBest2_345 Ca2+-bound open state

EntireName: bBest2_345 Ca2+-bound open state
Components
  • Complex: bBest2_345 Ca2+-bound open state
    • Protein or peptide: Bestrophin
  • Ligand: CALCIUM ION
  • 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 ION
  • Ligand: water

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Supramolecule #1: bBest2_345 Ca2+-bound open state

SupramoleculeName: bBest2_345 Ca2+-bound open state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Bos taurus (domestic cattle)
Molecular weightTheoretical: 210 KDa

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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 (domestic cattle)
Molecular weightTheoretical: 40.408918 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 YWDAAEARA

UniProtKB: Bestrophin-2

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

MacromoleculeName: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 3 / Number of copies: 50 / 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 #4: 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: 4 / 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 #5: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 600 / 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

Concentration5 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
200.0 mMNaClsodium 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
DetailsbBest2_345 Ca2+-bound open state

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 3203 / Average electron dose: 58.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1070653
Startup modelType of model: OTHER / Details: Ab initio
Final reconstructionApplied symmetry - Point group: C5 (5 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 1.93 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 117555
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelPDB ID:

6vx7


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8d1n:
bBest2_345 Ca2+-bound open state

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