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- PDB-6qp6: Cryo-EM structure of calcium-bound mTMEM16F lipid scramblase in d... -

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Basic information

Entry
Database: PDB / ID: 6qp6
TitleCryo-EM structure of calcium-bound mTMEM16F lipid scramblase in digitonin
ComponentsAnoctamin-6Calcium-dependent chloride channel
KeywordsMEMBRANE PROTEIN / lipid scrambles / TMEM16
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
1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Anoctamin-6
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsAlvadia, C. / Lim, N.K. / Clerico Mosina, V. / Oostergetel, G.T. / Dutzler, R. / Paulino, C.
Funding support Switzerland, Netherlands, 2items
OrganizationGrant numberCountry
European Research Council339116, AnoBest. Switzerland
Netherlands Organisation for Scientific Research740.018.016 Netherlands
CitationJournal: Elife / Year: 2019
Title: Cryo-EM structures and functional characterization of the murine lipid scramblase TMEM16F.
Authors: Carolina Alvadia / Novandy K Lim / Vanessa Clerico Mosina / Gert T Oostergetel / Raimund Dutzler / Cristina Paulino /
Abstract: The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium- ...The lipid scramblase TMEM16F initiates blood coagulation by catalyzing the exposure of phosphatidylserine in platelets. The protein is part of a family of membrane proteins, which encompasses calcium-activated channels for ions and lipids. Here, we reveal features of murine TMEM16F (mTMEM16F) that underlie its function as a lipid scramblase and an ion channel. The cryo-EM data of mTMEM16F in absence and presence of Ca define the ligand-free closed conformation of the protein and the structure of a Ca-bound intermediate. Both conformations resemble their counterparts of the scrambling-incompetent anion channel mTMEM16A, yet with distinct differences in the region of ion and lipid permeation. In conjunction with functional data, we demonstrate the relationship between ion conduction and lipid scrambling. Although activated by a common mechanism, both functions appear to be mediated by alternate protein conformations that are at equilibrium in the ligand-bound state.
History
DepositionFeb 13, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 6, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 3, 2019Group: Data collection / Source and taxonomy
Category: database_PDB_rev / database_PDB_rev_record ...database_PDB_rev / database_PDB_rev_record / em_admin / entity_src_gen / pdbx_database_proc
Item: _em_admin.last_update / _entity_src_gen.pdbx_host_org_cell_line

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)214,10910
Polymers212,7352
Non-polymers1,3748
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4740 Å2
ΔGint-63 kcal/mol
Surface area66660 Å2
MethodPISA

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Components

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


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 / Cell line (production host): HEK293T / Production host: Homo sapiens (human) / References: UniProt: Q6P9J9
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-P1O / 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE


Mass: 566.728 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C28H57NO8P / Feature type: SUBJECT OF INVESTIGATION / Comment: DDPC, phospholipid*YM

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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: mTMEM16F / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.212 MDa / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293T cells
Buffer solutionpH: 7.5
Details: 0.1% digitonin, 150 mM NaCl, 20 mM HEPES, pH 7.5 and 2 mM EGTA. 1 mM CaCl2 were added before freezing.
SpecimenConc.: 3.3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K

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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 FIELDBright-field microscopy / Nominal magnification: 49407 X / Calibrated magnification: 49407 X / Nominal defocus max: 3000 nm / Nominal defocus min: 300 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 3000 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): 105 K / Temperature (min): 90 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 7 / Num. of real images: 5633
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1bparticle selection
2EPU1.9.1image acquisition
4CTFFIND4.1.8CTF correction
7Cootmodel fitting
9RELION2.1binitial Euler assignment
10RELION3final Euler assignment
12RELION33D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1348247
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 219302 / Algorithm: BACK PROJECTION / Num. of class averages: 9 / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingPDB-ID: 5YOB

5yob

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