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- PDB-6p47: Cryo-EM structure of TMEM16F in digitonin without calcium -

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Basic information

Entry
Database: PDB / ID: 6p47
TitleCryo-EM structure of TMEM16F in digitonin without calcium
ComponentsAnoctamin-6Calcium-dependent chloride channel
KeywordsMEMBRANE PROTEIN / TMEM16F / scramblase
Function / homology
Function and homology information


calcium activated phospholipid scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / calcium activated galactosylceramide scrambling / phosphatidylserine exposure on blood platelet / positive regulation of potassium ion export across plasma membrane / purinergic nucleotide receptor signaling pathway / positive regulation of monoatomic ion transmembrane transport / phospholipid scramblase activity / activation of blood coagulation via clotting cascade ...calcium activated phospholipid scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / calcium activated galactosylceramide scrambling / phosphatidylserine exposure on blood platelet / positive regulation of potassium ion export across plasma membrane / purinergic nucleotide receptor signaling pathway / positive regulation of monoatomic ion transmembrane transport / phospholipid scramblase activity / activation of blood coagulation via clotting cascade / negative regulation of cell volume / intracellularly calcium-gated chloride channel activity / bone mineralization involved in bone maturation / pore complex assembly / cholinergic synapse / plasma membrane phospholipid scrambling / voltage-gated monoatomic ion channel activity / bleb assembly / positive regulation of phagocytosis, engulfment / Stimuli-sensing channels / voltage-gated chloride channel activity / calcium-activated cation channel activity / positive regulation of endothelial cell apoptotic process / positive regulation of monocyte chemotaxis / dendritic cell chemotaxis / chloride transport / phospholipid translocation / chloride channel activity / chloride channel complex / monoatomic cation transport / sodium ion transmembrane transport / regulation of postsynaptic membrane potential / positive regulation of bone mineralization / chloride transmembrane transport / Neutrophil degranulation / synaptic membrane / establishment of localization in cell / calcium ion transmembrane transport / blood coagulation / positive regulation of apoptotic process / protein homodimerization activity / identical protein binding / metal ion binding / plasma membrane / cytosol
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: 3.9 Å
AuthorsFeng, S. / Dang, S. / Han, T.W. / Ye, W. / Jin, P. / Cheng, T. / Li, J. / Jan, Y.N. / Jan, L.Y. / Cheng, Y.
Funding support United States, France, 6items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM098672 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)R35NS097229 United States
National Institutes of Health/Office of the DirectorS10OD0020054 United States
National Institutes of Health/Office of the DirectorS10OD021741 United States
Human Frontier Science Program (HFSP) France
CitationJournal: Cell Rep / Year: 2019
Title: Cryo-EM Studies of TMEM16F Calcium-Activated Ion Channel Suggest Features Important for Lipid Scrambling.
Authors: Shengjie Feng / Shangyu Dang / Tina Wei Han / Wenlei Ye / Peng Jin / Tong Cheng / Junrui Li / Yuh Nung Jan / Lily Yeh Jan / Yifan Cheng /
Abstract: As a Ca-activated lipid scramblase and ion channel that mediates Ca influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How ...As a Ca-activated lipid scramblase and ion channel that mediates Ca influx, TMEM16F relies on both functions to facilitate extracellular vesicle generation, blood coagulation, and bone formation. How a bona fide ion channel scrambles lipids remains elusive. Our structural analyses revealed the coexistence of an intact channel pore and PIP-dependent protein conformation changes leading to membrane distortion. Correlated to the extent of membrane distortion, many tightly bound lipids are slanted. Structure-based mutagenesis studies further reveal that neutralization of some lipid-binding residues or those near membrane distortion specifically alters the onset of lipid scrambling, but not Ca influx, thus identifying features outside of channel pore that are important for lipid scrambling. Together, our studies demonstrate that membrane distortion does not require open hydrophilic grooves facing the membrane interior and provide further evidence to suggest separate pathways for lipid scrambling and ion permeation.
History
DepositionMay 26, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 24, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / 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] / _cell.Z_PDB / _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: Anoctamin-6
B: Anoctamin-6


Theoretical massNumber of molelcules
Total (without water)212,7352
Polymers212,7352
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1900 Å2
ΔGint-17 kcal/mol
Surface area73850 Å2

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Components

#1: Protein Anoctamin-6 / Calcium-dependent chloride channel / Small-conductance calcium-activated nonselective cation channel / SCAN channel / Transmembrane ...Small-conductance calcium-activated nonselective cation channel / SCAN channel / Transmembrane protein 16F / TMEM16F


Mass: 106367.727 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Ano6, Tmem16f / Production host: Homo sapiens (human) / References: UniProt: Q6P9J9

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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: TMEM16F without calcium bound / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPES1
2150 mMsodium chlorideNaClSodium chloride1
31 mMEGTA1
40.06 %digitonin1
SpecimenEmbedding 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 IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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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 / 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: 12 sec. / Electron dose: 72 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2249

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1cisTEMparticle selection
2SerialEM3.7image acquisition
4CTFFINDCTF correction
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
11RELIONclassification
12RELION3D reconstruction
19PHENIXmodel refinement
20Cootmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1182627 / Details: automatically picked particles
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 324624 / Algorithm: BACK PROJECTION / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6P46

6p46


Pdb chain-ID: A

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