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- PDB-5nl2: cryo-EM structure of the mTMEM16A ion channel at 6.6 A resolution. -

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Basic information

Entry
Database: PDB / ID: 5nl2
Titlecryo-EM structure of the mTMEM16A ion channel at 6.6 A resolution.
ComponentsAnoctamin-1Calcium-dependent chloride channel
KeywordsMEMBRANE PROTEIN / TMEM16 family / ion channel / cryo-EM
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 / 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 / mucus secretion / cellular response to peptide / 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: 6.6 Å
AuthorsPaulino, C. / Neldner, Y. / Lam, K.M. / Kalienkova, V. / Brunner, J.D. / Schenck, S. / Dutzler, R.
Funding support Switzerland, 2items
OrganizationGrant numberCountry
European Research Council339116 Switzerland
University of ZurichForschungskredit FK-16-036 Switzerland
CitationJournal: Elife / Year: 2017
Title: Structural basis for anion conduction in the calcium-activated chloride channel TMEM16A.
Authors: Cristina Paulino / Yvonne Neldner / Andy Km Lam / Valeria Kalienkova / Janine Denise Brunner / Stephan Schenck / Raimund Dutzler /
Abstract: The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has ...The calcium-activated chloride channel TMEM16A is a member of a conserved protein family that comprises ion channels and lipid scramblases. Although the structure of the scramblase nhTMEM16 has defined the architecture of the family, it was unknown how a channel has adapted to cope with its distinct functional properties. Here we have addressed this question by the structure determination of mouse TMEM16A by cryo-electron microscopy and a complementary functional characterization. The protein shows a similar organization to nhTMEM16, except for changes at the site of catalysis. There, the conformation of transmembrane helices constituting a membrane-spanning furrow that provides a path for lipids in scramblases has changed to form an enclosed aqueous pore that is largely shielded from the membrane. Our study thus reveals the structural basis of anion conduction in a TMEM16 channel and it defines the foundation for the diverse functional behavior in the TMEM16 family.
History
DepositionApr 3, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jun 7, 2017Provider: repository / Type: Initial release
Revision 1.1Aug 30, 2017Group: Author supporting evidence / Data collection
Category: em_imaging_optics / em_software / pdbx_audit_support
Item: _em_imaging_optics.energyfilter_name / _em_software.name / _pdbx_audit_support.funding_organization
Revision 1.2Feb 28, 2018Group: Source and taxonomy / Category: entity_src_gen
Item: _entity_src_gen.pdbx_host_org_cell_line / _entity_src_gen.pdbx_host_org_strain

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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)222,1182
Polymers222,1182
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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


Mass: 111058.992 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ano1, Tmem16a
Cell line (production host): HEK293 stable mTMEM16A cell line (Flp-In System)
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 / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.110916 MDa / Experimental value: YES
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Strain: HEK293 / Cell: stabel mTMEM16A cell line (Flp-In System)
Buffer solutionpH: 7.5 / Details: 20 mM Hepes 150 mM NaCl 0.5 mM CaCl2 <0.12% digitonin
Buffer componentConc.: 20 mM / Name: Hepes
SpecimenConc.: 3.08 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: full-length (wild-type isoform ac) deglycosylated mTMEM16A
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: 2.5 ul sample volume 1-5 sec blotting time

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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 / Nominal magnification: 37037 X / Calibrated magnification: 37037 X / Nominal defocus max: 3800 nm / Nominal defocus min: 500 nm / Calibrated defocus min: 500 nm / Calibrated defocus max: 3800 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 / Temperature (max): 100 K / Temperature (min): 80 K
Image recordingAverage exposure time: 15 sec. / Electron dose: 80 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 5 / Num. of real images: 5503
Details: Data were collected in an automated fashion on a K2 Summit detector (Gatan) set to super-resolution mode with a pixel size of 0.675 A and a defocus range of -0.5 to -3.8 um using SerialEM. ...Details: Data were collected in an automated fashion on a K2 Summit detector (Gatan) set to super-resolution mode with a pixel size of 0.675 A and a defocus range of -0.5 to -3.8 um using SerialEM. Images were recorded for 15 sec with an initial sub-frame exposure time of 300 ms (50 frames total) with a dose of 1.5 e-/A^2/frame, and later with a sub-frame exposure time of 150 ms (100 frames total) with a dose of 0.8 e-/A^2/frame, resulting in a total accumulated dose on the specimen level of approximately 80 e-/A^2.
EM imaging opticsEnergyfilter name: GIF / Energyfilter upper: 10 eV / Energyfilter lower: -10 eV
Image scansSampling size: 5 µm / Width: 7420 / Height: 7676 / Movie frames/image: 50 / Used frames/image: 1-50

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2particle selection
2SerialEMimage acquisition
4CTFFIND4.1.5CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION2initial Euler assignment
11RELION2final Euler assignment
12RELION2classification
13RELION23D reconstruction
Image processingDetails: Fourier cropping (final pixel size 1.35 A), motion correction and dose-weighting of frames were performed by MotionCor2
CTF correctionDetails: The contrast transfer function (CTF) parameters were estimated on the movie frames by ctffind4.1
Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 755348
Details: Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, resulting in 4,178 images used for further analysis. Image processing was performed using ...Details: Images showing a strong drift, higher defocus than -3.8 um or a bad CTF estimation were discarded, resulting in 4,178 images used for further analysis. Image processing was performed using the software package RELION1.4 and at a later stage RELION2.0. Approximately 4,000 particles were manually picked to generate templates for automated particle selection. Following automated picking in RELION, false positives were eliminated manually or through a first round of 2D classification resulting in 755,348 particles.
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 6.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 213243 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT
Atomic model buildingPDB-ID: 4WIT

4wit

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0044282
ELECTRON MICROSCOPYf_angle_d0.6325930
ELECTRON MICROSCOPYf_dihedral_angle_d4.2692518
ELECTRON MICROSCOPYf_chiral_restr0.042840
ELECTRON MICROSCOPYf_plane_restr0.005838

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