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- PDB-8d1h: hBest2 Ca2+-unbound closed state -

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Basic information

Entry
Database: PDB / ID: 8d1h
TitlehBest2 Ca2+-unbound closed state
ComponentsBestrophin-2
KeywordsTRANSPORT PROTEIN / Ion channel / chloride channel / anion channel / pentamer
Function / homology
Function and homology information


intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / bicarbonate channel activity / chloride channel activity / ligand-gated monoatomic cation channel activity / membrane depolarization / chloride channel complex / cilium / Stimuli-sensing channels / sensory perception of smell ...intracellularly ligand-gated monoatomic ion channel activity / ligand-gated monoatomic anion channel activity / bicarbonate channel activity / chloride channel activity / ligand-gated monoatomic cation channel activity / membrane depolarization / chloride channel complex / cilium / Stimuli-sensing channels / sensory perception of smell / basolateral plasma membrane / metal ion binding / plasma membrane
Similarity search - Function
Bestrophin / Bestrophin/UPF0187 / Bestrophin, RFP-TM, chloride channel
Similarity search - Domain/homology
Chem-DU0 / 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE / Bestrophin-2a
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 1.94 Å
AuthorsOwji, A.P. / Kittredge, A. / Hendrickson, W.A. / Tingting, Y.
Funding support United States, 3items
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, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 13, 2022Provider: repository / Type: Initial release
Revision 1.1Feb 14, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
E: Bestrophin-2
B: Bestrophin-2
A: Bestrophin-2
C: Bestrophin-2
D: Bestrophin-2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)267,11156
Polymers233,9845
Non-polymers33,12651
Water10,899605
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Bestrophin-2 / Vitelliform macular dystrophy 2-like protein 1


Mass: 46796.898 Da / Num. of mol.: 5
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: BEST2, VMD2L1 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q8NFU1
#2: Chemical...
ChemComp-MC3 / 1,2-DIMYRISTOYL-RAC-GLYCERO-3-PHOSPHOCHOLINE


Mass: 677.933 Da / Num. of mol.: 45 / Source method: obtained synthetically / Formula: C36H72NO8P / Comment: phospholipid*YM
#3: Chemical
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


Mass: 516.752 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C32H52O5
#4: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 605 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: hBest2 Ca2+-unbound closed state / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.234 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293F
Buffer solutionpH: 7.8
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaCl1
240 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)1
30.005 %glyco-diosgenin1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: hBest2 Ca2+-unbound closed state
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 283.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm
Image recordingElectron dose: 58 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 3446
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2Leginonimage acquisition
4cryoSPARCCTF correction
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
14Cootmodel refinement
15REFMACmodel refinement
CTF correctionType: NONE
Particle selectionNum. of particles selected: 2853381
SymmetryPoint symmetry: C5 (5 fold cyclic)
3D reconstructionResolution: 1.94 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 656860 / Symmetry type: POINT

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