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- PDB-8b8k: Cryo-EM structure of Ca2+-bound mTMEM16F N562A mutant in Digitoni... -

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Basic information

Entry
Database: PDB / ID: 8b8k
TitleCryo-EM structure of Ca2+-bound mTMEM16F N562A mutant in Digitonin closed/closed
ComponentsAnoctamin-6Calcium-dependent chloride channel
KeywordsMEMBRANE PROTEIN / Lipid Transport / Lipid Scramblase / Ion Channel / Blood Coagulation / Membrane Fusion
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 / bleb assembly / voltage-gated monoatomic ion channel activity / 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 / phospholipid translocation / chloride transport / 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 / Anoctamin, dimerisation domain / 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: 3.01 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

Downloads & links

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)228,21510
Polymers226,8412
Non-polymers1,3748
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area4680 Å2
ΔGint-62 kcal/mol
Surface area66650 Å2

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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: 113420.602 Da / Num. of mol.: 2 / Mutation: N562A
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
#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 / Comment: DDPC, phospholipid*YM
Has ligand of interestY

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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 Ca2+-bound / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5 / Details: 20mM HEPES 150mM NaCl 2mM EGTA 0.1% Digitonin
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 / Details: 2mM free Ca2+ added shortly before freezing.

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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 defocus max: 2400 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 62.4 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: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 239346 / Symmetry type: POINT
Atomic model buildingB value: 97.7 / Space: REAL
Atomic model buildingPDB-ID: 6QP6

6qp6

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00312646
ELECTRON MICROSCOPYf_angle_d0.47517118
ELECTRON MICROSCOPYf_dihedral_angle_d11.184620
ELECTRON MICROSCOPYf_chiral_restr0.0391854
ELECTRON MICROSCOPYf_plane_restr0.0042118

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