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- PDB-8b8g: Cryo-EM structure of Ca2+-free mTMEM16F F518H mutant in Digitonin -

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Basic information

Entry
Database: PDB / ID: 8b8g
TitleCryo-EM structure of Ca2+-free mTMEM16F F518H mutant in Digitonin
ComponentsAnoctamin-6
KeywordsMEMBRANE PROTEIN / Lipid Transport / Lipid Scrambling / Ion Channel / Plasma Membrane / Blood Clotting / Exocytosis / Membrane Fusion
Function / homology
Function and homology information


calcium activated phospholipid scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / calcium activated galactosylceramide scrambling / positive regulation of potassium ion export across plasma membrane / purinergic nucleotide receptor signaling pathway / positive regulation of monoatomic ion transmembrane transport / phospholipid scramblase activity / intracellularly calcium-gated chloride channel activity / bone mineralization involved in bone maturation ...calcium activated phospholipid scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / calcium activated galactosylceramide scrambling / positive regulation of potassium ion export across plasma membrane / purinergic nucleotide receptor signaling pathway / positive regulation of monoatomic ion transmembrane transport / phospholipid scramblase activity / intracellularly calcium-gated chloride channel activity / bone mineralization involved in bone maturation / negative regulation of cell volume / cholinergic synapse / voltage-gated monoatomic ion channel activity / plasma membrane phospholipid scrambling / positive regulation of phagocytosis, engulfment / bleb assembly / Stimuli-sensing channels / calcium-activated cation channel activity / positive regulation of monocyte chemotaxis / dendritic cell chemotaxis / chloride transport / phospholipid translocation / chloride channel activity / chloride channel complex / 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
Similarity search - Function
Anoctamin, dimerisation domain / Anoctamin, dimerisation domain / Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.39 Å
AuthorsArndt, M. / Alvadia, C. / Straub, M.S. / Clerico-Mosina, V. / Paulino, C. / Dutzler, R.
Funding supportEuropean Union, 1items
OrganizationGrant numberCountry
European Research Council (ERC)339116European Union
CitationJournal: Nat Commun / Year: 2022
Title: Structural basis for the activation of the lipid scramblase TMEM16F.
Authors: Melanie Arndt / Carolina Alvadia / Monique S Straub / Vanessa Clerico Mosina / Cristina Paulino / Raimund Dutzler /
Abstract: TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in ...TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in response to an increase in intracellular Ca. However, the mechanism of how the protein facilitates both processes has remained elusive. Here we investigate the basis for TMEM16F activation. In a screen of residues lining the proposed site of conduction, we identify mutants with strongly activating phenotype. Structures of these mutants determined herein by cryo-electron microscopy show major rearrangements leading to the exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. The concomitant opening of a pore promotes ion conduction in the same protein conformation. Our work has revealed a mechanism that is distinct for this branch of the family and that will aid the development of a specific pharmacology for a promising drug target.
History
DepositionOct 4, 2022Deposition site: PDBE / Processing site: PDBE
Revision 1.0Nov 16, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)226,9092
Polymers226,9092
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1800 Å2
ΔGint-15 kcal/mol
Surface area70110 Å2
MethodPISA

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Components

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


Mass: 113454.602 Da / Num. of mol.: 2 / Mutation: F518H
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: mTMEM16F F518H / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5 / Details: 20mM HEPES 150mM NaCl 2mM EGTA
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
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-PROPANE / Humidity: 100 % / Chamber temperature: 277 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 FIELD / Nominal defocus max: 2400 nm / Nominal defocus min: 1000 nm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 69.8 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM software
IDNameCategory
2EPUimage acquisition
7Cootmodel fitting
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.39 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 181753 / Symmetry type: POINT
Atomic model buildingB value: 112.9 / Space: REAL
Atomic model buildingPDB-ID: 6QP6

6qp6

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00412422
ELECTRON MICROSCOPYf_angle_d0.52716852
ELECTRON MICROSCOPYf_dihedral_angle_d13.7624510
ELECTRON MICROSCOPYf_chiral_restr0.041836
ELECTRON MICROSCOPYf_plane_restr0.0052098

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