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- EMDB-15916: Cryo-EM structure of Ca2+-bound mTMEM16F N562A mutant in Digitoni... -

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Basic information

Entry
Database: EMDB / ID: EMD-15916
TitleCryo-EM structure of Ca2+-bound mTMEM16F N562A mutant in Digitonin closed/closed
Map data
Sample
  • Complex: mTMEM16F Ca2+-bound
    • Protein or peptide: Anoctamin-6
  • Ligand: CALCIUM ION
  • Ligand: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE
KeywordsMembrane Protein / Lipid Transport / Lipid Scramblase / Ion Channel / Blood Coagulation / Membrane Fusion
Function / homology
Function and homology information


calcium activated phospholipid scrambling / calcium activated galactosylceramide scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / positive regulation of potassium ion export across plasma membrane / positive regulation of monoatomic ion transmembrane transport / purinergic nucleotide receptor signaling pathway / phospholipid scramblase activity / bone mineralization involved in bone maturation / cholinergic synapse ...calcium activated phospholipid scrambling / calcium activated galactosylceramide scrambling / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / positive regulation of potassium ion export across plasma membrane / positive regulation of monoatomic ion transmembrane transport / purinergic nucleotide receptor signaling pathway / phospholipid scramblase activity / bone mineralization involved in bone maturation / cholinergic synapse / intracellularly calcium-gated chloride channel activity / plasma membrane phospholipid scrambling / negative regulation of cell volume / voltage-gated monoatomic ion channel activity / positive regulation of phagocytosis, engulfment / bleb assembly / Stimuli-sensing channels / calcium-activated cation channel activity / positive regulation of monocyte chemotaxis / chloride transport / dendritic cell chemotaxis / phospholipid translocation / regulation of postsynaptic membrane potential / positive regulation of bone mineralization / chloride channel complex / Neutrophil degranulation / chloride transmembrane transport / synaptic membrane / establishment of localization in cell / calcium ion transmembrane transport / blood coagulation / positive regulation of apoptotic process / protein homodimerization activity / metal ion binding / identical protein 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsArndt M / Alvadia C / Straub MS / Clerico-Mosina V / Paulino C / Dutzler R
Funding supportEuropean Union, 1 items
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, 2022-
Header (metadata) releaseNov 16, 2022-
Map releaseNov 16, 2022-
UpdateNov 13, 2024-
Current statusNov 13, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_15916.png

Downloads & links

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Map

FileDownload / File: emd_15916.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.3 Å/pix.
x 260 pix.
= 338.52 Å		1.3 Å/pix.
x 260 pix.
= 338.52 Å		1.3 Å/pix.
x 260 pix.
= 338.52 Å

Surface

Projections

Slices (1/3)

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.302 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.149
Minimum - Maximum-0.41304365 - 0.9758797
Average (Standard dev.)0.0008665701 (±0.020399326)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions260260260
Spacing260260260
CellA=B=C: 338.52002 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_15916_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_15916_half_map_2.map
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : mTMEM16F Ca2+-bound

EntireName: mTMEM16F Ca2+-bound
Components
  • Complex: mTMEM16F Ca2+-bound
    • Protein or peptide: Anoctamin-6
  • Ligand: CALCIUM ION
  • Ligand: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

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Supramolecule #1: mTMEM16F Ca2+-bound

SupramoleculeName: mTMEM16F Ca2+-bound / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Anoctamin-6

MacromoleculeName: Anoctamin-6 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 113.420602 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MQMMTRKVLL NMELEEDDDE DGDIVLENFD QTIVCPTFGS LENQQDFRTP EFEEFNGKPD SLFFTDGQRR IDFILVYEDE SKKENNKKG TNEKQKRKRQ AYESNLICHG LQLEATRSVS DDKLVFVKVH APWEVLCTYA EIMHIKLPLK PNDLKTRSPF G NLNWFTKV ...String:
MQMMTRKVLL NMELEEDDDE DGDIVLENFD QTIVCPTFGS LENQQDFRTP EFEEFNGKPD SLFFTDGQRR IDFILVYEDE SKKENNKKG TNEKQKRKRQ AYESNLICHG LQLEATRSVS DDKLVFVKVH APWEVLCTYA EIMHIKLPLK PNDLKTRSPF G NLNWFTKV LRVNESVIKP EQEFFTAPFE KSRMNDFYIL DRDSFFNPAT RSRIVYFILS RVKYQVMNNV NKFGINRLVS SG IYKAAFP LHDCRFNYES EDISCPSERY LLYREWAHPR SIYKKQPLDL IRKYYGEKIG IYFAWLGYYT QMLLLAAVVG VAC FLYGYL DQDNCTWSKE VCDPDIGGQI LMCPQCDRLC PFWRLNITCE SSKKLCIFDS FGTLIFAVFM GVWVTLFLEF WKRR QAELE YEWDTVELQQ EEQARPEYEA QCNHVVINEI TQEEERIPFT TCGKCIRVTL CASAVFFWIL LIIASVIGII VYRLS VFIV FSTTLPKNPN GTDPIQKYLT PQMATSITAS IISFIIIMIL NTIYEKVAIM ITNFELPRTQ TDYENSLTMK MFLFQF VAY YSSCFYIAFF KGKFVGYPGD PVYLLGKYRS EECDPGGCLL ELTTQLTIIM GGKAIWNNIQ EVLLPWVMNL IGRYKRV SG SEKITPRWEQ DYHLQPMGKL GLFYEYLEMI IQFGFVTLFV ASFPLAPLLA LVNNILEIRV DAWKLTTQFR RMVPEKAQ D IGAWQPIMQG IAILAVVTNA MIIAFTSDMI PRLVYYWSFS IPPYGDHTYY TMDGYINNTL SVFNITDFKN TDKENPYIG LGNYTLCRYR DFRNPPGHPQ EYKHNIYYWH VIAAKLAFII VMEHIIYSVK FFISYAIPDV SKITKSKIKR EKYLTQKLLH ESHLKDLTK NMGIIAERIG GTVDNSVRPK LEALEVLFQG PQGTEQKLIS EEDLRGASMD EKTTGWRGGH VVEGLAGELE Q LRARLEHH PQGQREP

UniProtKB: Anoctamin-6

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Macromolecule #3: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 3 / Number of copies: 2 / Formula: P1O
Molecular weightTheoretical: 566.728 Da
Chemical component information

ChemComp-P1O:
1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / DDPC, phospholipid*YM

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5 / Details: 20mM HEPES 150mM NaCl 2mM EGTA 0.1% Digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 2mM free Ca2+ added shortly before freezing..

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 62.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.4 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 239346
Initial angle assignmentType: OTHER / Details: cryoSPARC
Final angle assignmentType: OTHER / Details: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6qp6


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Overall B value: 97.7
Output model

PDB-8b8k:
Cryo-EM structure of Ca2+-bound mTMEM16F N562A mutant in Digitonin closed/closed

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