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- EMDB-15959: Cryo-EM Structure of Ca2+-bound mTMEM16F F518A_Q623A mutant in GDN -

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Basic information

Entry
Database: EMDB / ID: EMD-15959
TitleCryo-EM Structure of Ca2+-bound mTMEM16F F518A_Q623A mutant in GDN
Map dataMap of mTMEM16F F158A_Q623A mutant in Digitonin
Sample
  • Complex: Homodimeric mTMEM16F F518A Q623A mutant bound to Ca2+
    • Protein or peptide: Anoctamin-6,mTMEM16F
  • Ligand: CALCIUM ION
KeywordsLipid Scramblase / Ion Channel / Membrane Protein / Blood Coagulation / Viral Entry / Cell 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 / positive regulation of monoatomic ion transmembrane transport / purinergic nucleotide receptor signaling pathway / phospholipid scramblase activity / bone mineralization involved in bone maturation / intracellularly calcium-gated chloride channel activity ...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 / positive regulation of monoatomic ion transmembrane transport / purinergic nucleotide receptor signaling pathway / phospholipid scramblase activity / bone mineralization involved in bone maturation / intracellularly calcium-gated chloride channel activity / cholinergic synapse / plasma membrane phospholipid scrambling / voltage-gated monoatomic ion channel activity / negative regulation of cell volume / 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 / chloride channel complex / regulation of postsynaptic membrane potential / positive regulation of bone mineralization / Neutrophil degranulation / chloride transmembrane transport / establishment of localization in cell / synaptic membrane / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.93 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)European 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 14, 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_15959.png

Downloads & links

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Map

FileDownload / File: emd_15959.map.gz / Format: CCP4 / Size: 67 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of mTMEM16F F158A_Q623A mutant in Digitonin
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)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.302 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.18
Minimum - Maximum-0.8133537 - 0.92395663
Average (Standard dev.)-0.0013047437 (±0.018768433)
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: Halfmap of mTMEM16F F158A Q623A mutant in Digitonin

Fileemd_15959_half_map_1.map
AnnotationHalfmap of mTMEM16F F158A_Q623A mutant in Digitonin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Halfmap of mTMEM16F F158A Q623A mutant in Digitonin

Fileemd_15959_half_map_2.map
AnnotationHalfmap of mTMEM16F F158A_Q623A mutant in Digitonin
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Homodimeric mTMEM16F F518A Q623A mutant bound to Ca2+

EntireName: Homodimeric mTMEM16F F518A Q623A mutant bound to Ca2+
Components
  • Complex: Homodimeric mTMEM16F F518A Q623A mutant bound to Ca2+
    • Protein or peptide: Anoctamin-6,mTMEM16F
  • Ligand: CALCIUM ION

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Supramolecule #1: Homodimeric mTMEM16F F518A Q623A mutant bound to Ca2+

SupramoleculeName: Homodimeric mTMEM16F F518A Q623A mutant bound to Ca2+ / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Anoctamin-6,mTMEM16F

MacromoleculeName: Anoctamin-6,mTMEM16F / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 113.330484 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 IISAIIIMIL NTIYEKVAIM ITNFELPRTQ TDYENSLTMK MFLFQF VNY YSSCFYIAFF KGKFVGYPGD PVYLLGKYRS EECDPGGCLL ELTTQLTIIM GGKAIWNNIA 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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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 0.03% GDN
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: 59.46 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / 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: 2.93 Å / Resolution method: FSC 0.143 CUT-OFF
Details: The map was masked for calculation the final resolution (cryoSPARC auto-masking)
Number images used: 183302
Initial angle assignmentType: OTHER / Details: cryoSPARC
Final angle assignmentType: OTHER / Details: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Overall B value: 73.9
Output model

PDB-8bc1:
Cryo-EM Structure of Ca2+-bound mTMEM16F F518A_Q623A mutant in GDN

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