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- EMDB-27137: hBest1_345 Ca2+-bound open state -

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Basic information

Entry
Database: EMDB / ID: EMD-27137
TitlehBest1_345 Ca2+-bound open state
Map data
Sample
  • Complex: hBest1_345 Ca2+-bound open state
    • Protein or peptide: Bestrophin-1
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: CALCIUM ION
  • Ligand: water
KeywordsIon channel / chloride channel / anion channel / pentamer / TRANSPORT PROTEIN
Function / homology
Function and homology information


membrane microdomain / bicarbonate channel activity / transepithelial chloride transport / gamma-aminobutyric acid secretion, neurotransmission / detection of light stimulus involved in visual perception / ligand-gated channel activity / intracellularly calcium-gated chloride channel activity / bicarbonate transmembrane transporter activity / glutamate secretion / chloride transport ...membrane microdomain / bicarbonate channel activity / transepithelial chloride transport / gamma-aminobutyric acid secretion, neurotransmission / detection of light stimulus involved in visual perception / ligand-gated channel activity / intracellularly calcium-gated chloride channel activity / bicarbonate transmembrane transporter activity / glutamate secretion / chloride transport / chloride channel activity / protein complex oligomerization / regulation of calcium ion transport / chloride channel complex / visual perception / basal plasma membrane / regulation of synaptic plasticity / Stimuli-sensing channels / presynapse / monoatomic ion transmembrane transport / basolateral plasma membrane / identical protein binding / membrane / plasma membrane / cytosol
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: 2.44 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_27137.png

Downloads & links

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Map

FileDownload / File: emd_27137.map.gz / Format: CCP4 / Size: 8.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 148 pix.
= 122.84 Å		0.83 Å/pix.
x 124 pix.
= 102.92 Å		0.83 Å/pix.
x 124 pix.
= 102.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.9
Minimum - Maximum-4.059393 - 6.648775
Average (Standard dev.)-0.021032 (±0.6105105)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin888762
Dimensions124124148
Spacing124124148
CellA: 102.92 Å / B: 102.92 Å / C: 122.84 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: full map

Fileemd_27137_additional_1.map
Annotationfull map
Projections & Slices
AxesZYX

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Half map: half map 1

Fileemd_27137_half_map_1.map
Annotationhalf map 1
Projections & Slices
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Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2

Fileemd_27137_half_map_2.map
Annotationhalf map 2
Projections & Slices
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Sample components

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Entire : hBest1_345 Ca2+-bound open state

EntireName: hBest1_345 Ca2+-bound open state
Components
  • Complex: hBest1_345 Ca2+-bound open state
    • Protein or peptide: Bestrophin-1
  • Ligand: 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: CALCIUM ION
  • Ligand: water

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Supramolecule #1: hBest1_345 Ca2+-bound open state

SupramoleculeName: hBest1_345 Ca2+-bound open state / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 338 KDa

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Macromolecule #1: Bestrophin-1

MacromoleculeName: Bestrophin-1 / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 40.60407 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MTITYTSQVA NARLGSFSRL LLCWRGSIYK LLYGEFLIFL LCYYIIRFIY RLALTEEQQL MFEKLTLYCD SYIQLIPISF VLGFYVTLV VTRWWNQYEN LPWPDRLMSL VSGFVEGKDE QGRLLRRTLI RYANLGNVLI LRSVSTAVYK RFPSAQHLVQ A GFMTPAEH ...String:
MTITYTSQVA NARLGSFSRL LLCWRGSIYK LLYGEFLIFL LCYYIIRFIY RLALTEEQQL MFEKLTLYCD SYIQLIPISF VLGFYVTLV VTRWWNQYEN LPWPDRLMSL VSGFVEGKDE QGRLLRRTLI RYANLGNVLI LRSVSTAVYK RFPSAQHLVQ A GFMTPAEH KQLEKLSLPH NMFWVPWVWF ANLSMKAWLG GRIRDPILLQ SLLNEMNTLR TQCGHLYAYD WISIPLVYTQ VV TVAVYSF FLTCLVGRQF LNPAKAYPGH ELDLVVPVFT FLQFFFYVGW LKVAEQLINP FGEDDDDFET NWIVDRNLQV SLL AVDEMH QDLPRMEPDM YWNKPEPQP

UniProtKB: Bestrophin-1

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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: 40 / 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: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 3 / Number of copies: 5 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #4: water

MacromoleculeName: water / type: ligand / ID: 4 / Number of copies: 230 / 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
DetailshBest1_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: 2138 / 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: 803036
Startup modelType of model: OTHER / Details: Ab initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.44 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 25050
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC

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