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- PDB-7b5d: Structure of calcium-free mTMEM16A(ac)-I551A chloride channel at ... -

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Basic information

Entry
Database: PDB / ID: 7b5d
TitleStructure of calcium-free mTMEM16A(ac)-I551A chloride channel at 3.3 A resolution
ComponentsAnoctamin-1
KeywordsMEMBRANE PROTEIN / ligand-gated ion channel / anoctamin-1
Function / homology
Function and homology information


glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / voltage-gated chloride channel activity / Stimuli-sensing channels / chloride transport ...glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / mucus secretion / cellular response to peptide / voltage-gated chloride channel activity / Stimuli-sensing channels / chloride transport / chloride channel activity / positive regulation of insulin secretion involved in cellular response to glucose stimulus / detection of temperature stimulus involved in sensory perception of pain / chloride channel complex / chloride transmembrane transport / cell projection / establishment of localization in cell / regulation of membrane potential / presynaptic membrane / phospholipase C-activating G protein-coupled receptor signaling pathway / cellular response to heat / apical plasma membrane / external side of plasma membrane / signaling receptor binding / glutamatergic synapse / protein homodimerization activity / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Anoctamin, dimerisation domain / Anoctamin, dimerisation domain / Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsLam, A.K.M. / Rheinberger, J. / Paulino, C. / Dutzler, R.
Funding support Switzerland, Netherlands, 4items
OrganizationGrant numberCountry
European Research Council (ERC)339116 Switzerland
Netherlands Organisation for Scientific Research (NWO)722.017.001 Netherlands
Netherlands Organisation for Scientific Research (NWO)740.018.016 Netherlands
University of ZurichFK-18-048 Switzerland
CitationJournal: Nat Commun / Year: 2021
Title: Gating the pore of the calcium-activated chloride channel TMEM16A.
Authors: Andy K M Lam / Jan Rheinberger / Cristina Paulino / Raimund Dutzler /
Abstract: The binding of cytoplasmic Ca to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an ...The binding of cytoplasmic Ca to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca.
History
DepositionDec 3, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Feb 10, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 17, 2021Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
A: Anoctamin-1
B: Anoctamin-1


Theoretical massNumber of molelcules
Total (without water)222,0342
Polymers222,0342
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1910 Å2
ΔGint-9 kcal/mol
Surface area69960 Å2
MethodPISA

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Components

#1: Protein Anoctamin-1 / Transmembrane protein 16A


Mass: 111016.914 Da / Num. of mol.: 2 / Mutation: I551A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ano1, Tmem16a / Production host: Homo sapiens (human) / References: UniProt: Q8BHY3

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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: mTMEM16A(ac)-I551A in calcium-free form / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.221748 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Buffer component
IDConc.NameBuffer-ID
1150 mMNaCl1
22 mMEGTA1
320 mMHEPES1
40.01 %GDN1
51.5 mMdiC8-PI(4,5)P21
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 49407 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 53 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

Software
NameVersionClassificationNB
phenix.real_space_refine1.16_3549refinement
PHENIX1.16_3549refinement
EM software
IDNameVersionCategory
1crYOLO1.4.1particle selection
2EPU2.4image acquisition
4CTFFIND4.1.13CTF correction
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.16model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 138320 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 5OYG

5oyg

RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 27.31 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.005111752
ELECTRON MICROSCOPYf_angle_d0.811815908
ELECTRON MICROSCOPYf_chiral_restr0.04941752
ELECTRON MICROSCOPYf_plane_restr0.00691998
ELECTRON MICROSCOPYf_dihedral_angle_d9.25686998

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