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- PDB-6bgj: Cryo-EM structure of the TMEM16A calcium-activated chloride chann... -

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Basic information

Entry
Database: PDB / ID: 6bgj
TitleCryo-EM structure of the TMEM16A calcium-activated chloride channel in LMNG
ComponentsAnoctamin-1Calcium-dependent chloride channel
KeywordsMEMBRANE PROTEIN / Chloride channel / TMEM16 family
Function / homology
Function and homology information


glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / cellular response to peptide / mucus secretion / Stimuli-sensing channels / voltage-gated chloride channel activity / calcium-activated cation channel activity ...glial cell projection elongation / trachea development / iodide transmembrane transporter activity / iodide transport / intracellularly calcium-gated chloride channel activity / cellular response to peptide / mucus secretion / Stimuli-sensing channels / voltage-gated chloride channel activity / calcium-activated cation channel activity / protein localization to membrane / 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 / monoatomic cation transport / chloride transmembrane transport / regulation of membrane potential / cell projection / establishment of localization in cell / presynaptic membrane / cellular response to heat / phospholipase C-activating G protein-coupled receptor signaling pathway / apical plasma membrane / external side of plasma membrane / signaling receptor binding / glutamatergic synapse / protein homodimerization activity / nucleoplasm / identical protein binding / metal ion binding / plasma membrane
Similarity search - Function
Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsDang, S. / Feng, S. / Tien, J. / Peters, C.J. / Bulkley, D. / Lolicato, M. / Zhao, J. / Zuberbuhler, K. / Ye, W. / Qi, L. ...Dang, S. / Feng, S. / Tien, J. / Peters, C.J. / Bulkley, D. / Lolicato, M. / Zhao, J. / Zuberbuhler, K. / Ye, W. / Qi, L. / Chen, T. / Craik, C.S. / Jan, Y.N. / Minor Jr., D.L. / Cheng, Y. / Jan, L.Y.
Funding support United States, 9items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM098672 United States
National Institutes of Health/Office of the DirectorS100D0020054 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R01NS069229 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)R35NS097227 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL080050 United States
National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD)R01DC007664 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)CA196276 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM111126 United States
National Institutes of Health/National Institute on Drug Abuse (NIH/NIDA)K99DA041500 United States
CitationJournal: Nature / Year: 2017
Title: Cryo-EM structures of the TMEM16A calcium-activated chloride channel.
Authors: Shangyu Dang / Shengjie Feng / Jason Tien / Christian J Peters / David Bulkley / Marco Lolicato / Jianhua Zhao / Kathrin Zuberbühler / Wenlei Ye / Lijun Qi / Tingxu Chen / Charles S Craik / ...Authors: Shangyu Dang / Shengjie Feng / Jason Tien / Christian J Peters / David Bulkley / Marco Lolicato / Jianhua Zhao / Kathrin Zuberbühler / Wenlei Ye / Lijun Qi / Tingxu Chen / Charles S Craik / Yuh Nung Jan / Daniel L Minor / Yifan Cheng / Lily Yeh Jan /
Abstract: Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the ...Calcium-activated chloride channels (CaCCs) encoded by TMEM16A control neuronal signalling, smooth muscle contraction, airway and exocrine gland secretion, and rhythmic movements of the gastrointestinal system. To understand how CaCCs mediate and control anion permeation to fulfil these physiological functions, knowledge of the mammalian TMEM16A structure and identification of its pore-lining residues are essential. TMEM16A forms a dimer with two pores. Previous CaCC structural analyses have relied on homology modelling of a homologue (nhTMEM16) from the fungus Nectria haematococca that functions primarily as a lipid scramblase, as well as subnanometre-resolution electron cryo-microscopy. Here we present de novo atomic structures of the transmembrane domains of mouse TMEM16A in nanodiscs and in lauryl maltose neopentyl glycol as determined by single-particle electron cryo-microscopy. These structures reveal the ion permeation pore and represent different functional states. The structure in lauryl maltose neopentyl glycol has one Ca ion resolved within each monomer with a constricted pore; this is likely to correspond to a closed state, because a CaCC with a single Ca occupancy requires membrane depolarization in order to open (C.J.P. et al., manuscript submitted). The structure in nanodiscs has two Ca ions per monomer and its pore is in a closed conformation; this probably reflects channel rundown, which is the gradual loss of channel activity that follows prolonged CaCC activation in 1 mM Ca. Our mutagenesis and electrophysiological studies, prompted by analyses of the structures, identified ten residues distributed along the pore that interact with permeant anions and affect anion selectivity, as well as seven pore-lining residues that cluster near pore constrictions and regulate channel gating. Together, these results clarify the basis of CaCC anion conduction.
History
DepositionOct 28, 2017Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 27, 2017Provider: repository / Type: Initial release
Revision 1.1Jan 10, 2018Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Jan 17, 2018Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.3Feb 14, 2018Group: Other / Category: cell / Item: _cell.length_a / _cell.length_b / _cell.length_c
Revision 1.4Apr 11, 2018Group: Data collection / Other / Structure summary / Category: audit_author / cell / Item: _audit_author.name / _cell.Z_PDB
Revision 1.5Dec 4, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.6Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _pdbx_audit_support.funding_organization
Revision 1.7Mar 13, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)211,2884
Polymers211,2082
Non-polymers802
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area1410 Å2
ΔGint-33 kcal/mol
Surface area47930 Å2

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Components

#1: Protein Anoctamin-1 / Calcium-dependent chloride channel / Transmembrane protein 16A


Mass: 105603.984 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Calcium ions / Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ano1, Tmem16a / Production host: Homo sapiens (human) / References: UniProt: Q8BHY3
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca

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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: TMEM16A Calcium-activated chloride channel / Type: CELL / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 9
Buffer component
IDConc.NameFormulaBuffer-ID
110 mMtris nitrateTrisNO31
2150 mMpotassium nitrateKNO31
31 mMcalcium chlorideCaCl21
40.02 mMLauryl Maltose Neopentyl GlycolLMNG1
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: blot 6-8 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Calibrated magnification: 29000 X / Calibrated defocus min: 800 nm / Calibrated defocus max: 1900 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7882
Details: 5326 images for TMEM16A alone in LMNG, 2556 images for TMEM16A with Fab bound in LMNG
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.12_2829: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatch0.56particle selection
2SerialEM3.7betaimage acquisition
4Gctf0.1.06CTF correction
10RELION2.1beta1initial Euler assignment
11RELION2.1beta1final Euler assignment
12RELION2.1beta1classification
13RELION2.1beta3D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 927414
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 251851 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0087609
ELECTRON MICROSCOPYf_angle_d1.18410370
ELECTRON MICROSCOPYf_dihedral_angle_d6.3124424
ELECTRON MICROSCOPYf_chiral_restr0.0611207
ELECTRON MICROSCOPYf_plane_restr0.011301

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