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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-1
KeywordsMEMBRANE PROTEIN / Chloride channel / TMEM16 family
Function/homologyAnoctamin-1 / Anoctamin, dimerisation domain / iodide transmembrane transporter activity / iodide transport / intracellular calcium activated chloride channel activity / Anoctamin / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Calcium-activated chloride channel / trachea development / calcium activated cation channel activity ...Anoctamin-1 / Anoctamin, dimerisation domain / iodide transmembrane transporter activity / iodide transport / intracellular calcium activated chloride channel activity / Anoctamin / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Calcium-activated chloride channel / trachea development / calcium activated cation channel activity / Stimuli-sensing channels / voltage-gated chloride channel activity / chloride channel activity / chloride channel complex / detection of temperature stimulus involved in sensory perception of pain / chloride transport / chloride transmembrane transport / cation transport / positive regulation of insulin secretion involved in cellular response to glucose stimulus / phospholipase C-activating G-protein coupled receptor signaling pathway / regulation of membrane potential / cellular response to heat / apical plasma membrane / external side of plasma membrane / protein heterodimerization activity / protein homodimerization activity / plasma membrane / cytoplasm / Anoctamin-1
Function and homology information
Specimen sourceMus musculus / mammal / House mouse /
MethodElectron microscopy (3.8 Å resolution / Particle / Single particle) / Transmission electron microscopy
AuthorsDang, 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.
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 / 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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Oct 28, 2017 / Release: Dec 27, 2017
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 27, 2017Structure modelrepositoryInitial release
1.1Jan 10, 2018Structure modelDatabase referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last
1.2Jan 17, 2018Structure modelAuthor supporting evidencepdbx_audit_support_pdbx_audit_support.funding_organization
1.3Feb 14, 2018Structure modelOthercell_cell.length_a / _cell.length_b / _cell.length_c
1.4Apr 11, 2018Structure modelData collection / Other / Structure summaryaudit_author / cell_audit_author.name / _cell.Z_PDB

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)211,2884
Polyers211,2082
Non-polymers802
Water0
1


TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)1410
ΔGint (kcal/M)-33
Surface area (Å2)47930

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Components

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


Mass: 105603.984 Da / Num. of mol.: 2 / Details: Calcium ions / Source: (gene. exp.) Mus musculus / mammal / House mouse / / Gene: Ano1, Tmem16a / Production host: Homo sapiens / References: UniProt:Q8BHY3
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Formula: Ca / : Calcium

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: SINGLE PARTICLE

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Sample preparation

ComponentName: TMEM16A Calcium-activated chloride channel / Type: CELL / Entity ID: 1 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus
Source (recombinant)Organism: Homo sapiens
Buffer solutionpH: 9
Buffer component
IDConc.UnitsNameFormulaBuffer ID
110mMtris nitrateTrisNO31
2150mMpotassium nitrateKNO31
31mMcalcium chlorideCaCl21
40.02mMLauryl 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 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 kelvins / Details: blot 6-8 seconds before plunging

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated magnification: 29000 / Calibrated defocus min: 800 nm / Calibrated defocus max: 1900 nm / Cs: 2.7 mm / C2 aperture diameter: 100 mm / 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
Details: 5326 images for TMEM16A alone in LMNG, 2556 images for TMEM16A with Fab bound in LMNG
Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 2 / Number of real images: 7882
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
13RELION2.1betaRECONSTRUCTION
CTF correctionType: NONE
Particle selectionNumber of particles selected: 927414
SymmetryPoint symmetry: C2
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 251851 / Algorithm: BACK PROJECTION / Number of class averages: 2 / Symmetry type: POINT
Atomic model buildingRef protocol: AB INITIO MODEL / Ref 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_restr0.0101301

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