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- EMDB-4611: Cryo-EM structure of calcium-bound mTMEM16F lipid scramblase in d... -

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Basic information

Entry
Database: EMDB / ID: 4611
TitleCryo-EM structure of calcium-bound mTMEM16F lipid scramblase in digitonin
Map data
SamplemTMEM16F
  • Anoctamin-6Calcium-dependent chloride channel
  • (ligand) x 2
Function / homologyAnoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin-6 / Calcium-activated chloride channel / Neutrophil degranulation / Stimuli-sensing channels / Anoctamin / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / purinergic nucleotide receptor signaling pathway ...Anoctamin, dimerisation domain / Dimerisation domain of Ca+-activated chloride-channel, anoctamin / Anoctamin-6 / Calcium-activated chloride channel / Neutrophil degranulation / Stimuli-sensing channels / Anoctamin / calcium activated phosphatidylserine scrambling / calcium activated phosphatidylcholine scrambling / purinergic nucleotide receptor signaling pathway / calcium activated galactosylceramide scrambling / phosphatidylserine exposure on blood platelet / calcium activated phospholipid scrambling / positive regulation of potassium ion export across plasma membrane / phospholipid scramblase activity / plasma membrane phospholipid scrambling / intracellular calcium activated chloride channel activity / negative regulation of cell volume / positive regulation of ion transmembrane transport / bone mineralization involved in bone maturation / pore complex assembly / activation of blood coagulation via clotting cascade / calcium activated cation channel activity / voltage-gated chloride channel activity / bleb assembly / chloride transport / sodium ion transmembrane transport / cation transport / positive regulation of endothelial cell apoptotic process / voltage-gated ion channel activity / chloride channel complex / chloride transmembrane transport / positive regulation of bone mineralization / dendritic cell chemotaxis / positive regulation of phagocytosis, engulfment / positive regulation of monocyte chemotaxis / calcium ion transmembrane transport / blood coagulation / positive regulation of apoptotic process / protein homodimerization activity / plasma membrane / cytosol / Anoctamin-6
Function and homology information
SourceMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / 3.2 Å resolution
AuthorsAlvadia C / Lim NK / Clerico Mosina V / Oostergetel GT / Dutzler R / Paulino C
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 ...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.
Validation ReportPDB-ID: 6qp6

SummaryFull reportAbout validation report
DateDeposition: Feb 13, 2019 / Header (metadata) release: Mar 6, 2019 / Map release: Mar 6, 2019 / Last update: Mar 6, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6qp6
  • Surface level: 0.05
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_4611.map.gz (map file in CCP4 format, 42593 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
220 pix
1.01 Å/pix.
= 222.64 Å
220 pix
1.01 Å/pix.
= 222.64 Å
220 pix
1.01 Å/pix.
= 222.64 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.012 Å
Density
Contour Level:0.04 (by author), 0.05 (movie #1):
Minimum - Maximum0.0 - 0.9595831
Average (Standard dev.)0.03758431 (0.16969879)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions220220220
Origin0.00.00.0
Limit219.0219.0219.0
Spacing220220220
CellA=B=C: 222.64 Å
α=β=γ: 90.0 deg.

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
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.2820.3520.000

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Supplemental data

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Mask #1

Fileemd_4611_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire mTMEM16F

EntireName: mTMEM16F / Number of components: 4

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Component #1: protein, mTMEM16F

ProteinName: mTMEM16F / Recombinant expression: No
MassTheoretical: 212 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Homo sapiens (human) / Cell of expression system: HEK293T cells

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Component #2: protein, Anoctamin-6

ProteinName: Anoctamin-6Calcium-dependent chloride channel / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 106.367727 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Homo sapiens (human)

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Component #3: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Component #4: ligand, 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE

LigandName: 1,2-DIDECANOYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.566728 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 3.3 mg/ml
Buffer solution: 0.1% digitonin, 150 mM NaCl, 20 mM HEPES, pH 7.5 and 2 mM EGTA. 1 mM CaCl2 were added before freezing.
pH: 7.5
Support filmat 5 mA
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 288 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
ImagingMicroscope: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Electron dose: 52 e/Å2 / Illumination mode: FLOOD BEAM
LensMagnification: 49407.0 X (nominal), 49407.0 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 300.0 - 3000.0 nm / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: K ( 90.0 - 105.0 K)
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 5633

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 219302
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Input PDB model: 5YOB
Output model

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