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- PDB-6qpi: Cryo-EM structure of calcium-free mTMEM16F lipid scramblase in na... -

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Basic information

Entry
Database: PDB / ID: 6qpi
TitleCryo-EM structure of calcium-free mTMEM16F lipid scramblase in nanodisc
ComponentsAnoctamin-6Calcium-dependent chloride channel
KeywordsMEMBRANE PROTEIN / membrane protein / lipid scrambles / TMEM16
Function / homologyAnoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin-6 / Calcium-activated chloride channel / Neutrophil degranulation / Stimuli-sensing channels / Anoctamin / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / purinergic nucleotide receptor signaling pathway ...Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin-6 / Calcium-activated chloride channel / Neutrophil degranulation / Stimuli-sensing channels / Anoctamin / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / purinergic nucleotide receptor signaling pathway / calcium activated galactosylceramide scrambling / phosphatidylserine exposure on blood platelet / calcium activated phospholipid scrambling / positive regulation of potassium ion export across plasma membrane / phospholipid scramblase activity / plasma membrane phospholipid scrambling / intracellular calcium activated chloride channel activity / negative regulation of cell volume / positive regulation of ion transmembrane transport / bone mineralization involved in bone maturation / pore complex assembly / activation of blood coagulation via clotting cascade / calcium activated cation channel activity / voltage-gated chloride channel activity / bleb assembly / chloride transport / sodium ion transmembrane transport / cation transport / positive regulation of endothelial cell apoptotic process / voltage-gated ion channel activity / chloride channel complex / chloride transmembrane transport / positive regulation of bone mineralization / dendritic cell chemotaxis / positive regulation of phagocytosis, engulfment / positive regulation of monocyte chemotaxis / calcium ion transmembrane transport / blood coagulation / positive regulation of apoptotic process / protein homodimerization activity / plasma membrane / cytosol / Anoctamin-6
Function and homology information
Specimen sourceMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.3 Å resolution
AuthorsAlvadia, C. / Lim, N.K. / Clerico Mosina, V. / Oostergetel, G.T. / Dutzler, R. / Paulino, C.
Funding supportSwitzerland , Netherlands , 2 items
OrganizationGrant numberCountry
European Research Council339116, AnoBest.Switzerland
Netherlands Organisation for Scientific Research740.018.016Netherlands
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 ...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.
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Feb 14, 2019 / Release: Mar 6, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 6, 2019Structure modelrepositoryInitial release
1.1Mar 20, 2019Structure modelAuthor supporting evidence / Data collectionem_admin / em_single_particle_entity / pdbx_database_proc_em_admin.last_update / _em_single_particle_entity.point_symmetry

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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
Polyers212,7352
Non-polymers00
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide 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: (gene. exp.) Mus musculus (house mouse) / Gene: Ano6, Tmem16f / Cell line (production host): HEK293T / 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 / 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)Cell: HEK293T cells / Organism: Homo sapiens (human)
Buffer solutionDetails: 150 mM NaCl, 20 mM HEPES, pH 7.5 and 2 mM EGTA / pH: 7.5
SpecimenConc.: 1 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 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 kelvins

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyMicroscope model: 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 / Calibrated magnification: 49407 / 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 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 kelvins / Temperature (min): 90 kelvins
Image recordingAverage exposure time: 9 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 10 / Number of real images: 8706
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
9PHENIXmodel refinement
10RELION2.1binitial Euler assignment
11RELION2.1bfinal Euler assignment
13RELION2.1b3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNumber of particles selected: 1593115
SymmetryPoint symmetry: C2
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 280891 / Algorithm: BACK PROJECTION / Number of class averages: 9 / Symmetry type: POINT
Atomic model buildingRef space: REAL

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