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- EMDB-4614: Cryo-EM structure of calcium-free mTMEM16F lipid scramblase in na... -

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Basic information

Entry
Database: EMDB / ID: EMD-4614
TitleCryo-EM structure of calcium-free mTMEM16F lipid scramblase in nanodisc
Map dataNone
Sample
  • Complex: mTMEM16F
    • Protein or peptide: Anoctamin-6
Keywordsmembrane protein / lipid scrambles / TMEM16
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 / negative regulation of cell volume / 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 / purinergic nucleotide receptor signaling pathway / positive regulation of monoatomic ion transmembrane transport / phospholipid scramblase activity / negative regulation of cell volume / intracellularly calcium-gated chloride channel activity / bone mineralization involved in bone maturation / cholinergic synapse / plasma membrane phospholipid scrambling / bleb assembly / voltage-gated monoatomic ion channel activity / Stimuli-sensing channels / positive regulation of phagocytosis, engulfment / calcium-activated cation channel activity / positive regulation of monocyte chemotaxis / chloride transport / dendritic cell chemotaxis / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsAlvadia C / Lim NK
Funding support Switzerland, Netherlands, 2 items
OrganizationGrant numberCountry
European Research Council339116, AnoBest. Switzerland
Netherlands Organisation for Scientific Research740.018.016 Netherlands
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 calcium- ...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.
History
DepositionFeb 14, 2019-
Header (metadata) releaseMar 6, 2019-
Map releaseMar 6, 2019-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.074
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.074
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qpi
  • Surface level: 0.074
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4614.png

Downloads & links

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Map

FileDownload / File: emd_4614.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 1.012 Å
Density
Contour LevelBy AUTHOR: 0.074 / Movie #1: 0.074
Minimum - Maximum-0.42225942 - 0.5061744
Average (Standard dev.)0.0014710699 (±0.0138458945)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 222.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z222.640222.640222.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.4220.5060.001

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Supplemental data

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Mask #1

Fileemd_4614_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 used during refinement and for...

Fileemd_4614_half_map_1.map
AnnotationHalf map 1 used during refinement and for FSC gold-standard resolution calculation mTMEM16F_2N2_noCa.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 used during refinement and for...

Fileemd_4614_half_map_2.map
AnnotationHalf map 2 used during refinement and for FSC gold-standard resolution calculation mTMEM16F_2N2_noCa.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : mTMEM16F

EntireName: mTMEM16F
Components
  • Complex: mTMEM16F
    • Protein or peptide: Anoctamin-6

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Supramolecule #1: mTMEM16F

SupramoleculeName: mTMEM16F / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 212 KDa

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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: 106.367727 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 VNY 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 LE

UniProtKB: Anoctamin-6

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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

Concentration1 mg/mL
BufferpH: 7.5 / Details: 150 mM NaCl, 20 mM HEPES, pH 7.5 and 2 mM EGTA
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: at 5 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 90.0 K / Max: 105.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 10 / Number real images: 8706 / Average exposure time: 9.0 sec. / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 49407
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1593115
Startup modelType of model: PDB ENTRY
Final reconstructionNumber classes used: 9 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1b) / Number images used: 280891
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1b)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1b)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

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

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