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- EMDB-8931: afTMEM16 reconstituted in nanodiscs in the absence of Ca2+ -

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Entry
Database: EMDB / ID: 8931
TitleafTMEM16 reconstituted in nanodiscs in the absence of Ca2+
Map dataafTMEM16 reconstituted in nanodiscs in the absence of Ca2+
SampleafTMEM16 reconstituted in nanodiscs in the absence of Ca2+
  • Plasma membrane channel protein (Aqy1), putative
Function / homologyCalcium-activated chloride channel / Anoctamin / phospholipid scramblase activity / phospholipid translocation / voltage-gated calcium channel activity / ion transmembrane transport / integral component of membrane / Plasma membrane channel protein (Aqy1), putative
Function and homology information
SourceAspergillus fumigatus (mold) / Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) (mold)
Methodsingle particle reconstruction / cryo EM / 3.89 Å resolution
AuthorsFalzone ME / Accardi A
CitationJournal: Elife / Year: 2019
Title: Structural basis of Ca-dependent activation and lipid transport by a TMEM16 scramblase.
Authors: Maria E Falzone / Jan Rheinberger / Byoung-Cheol Lee / Thasin Peyear / Linda Sasset / Ashleigh M Raczkowski / Edward T Eng / Annarita Di Lorenzo / Olaf S Andersen / Crina M Nimigean / Alessio Accardi
Abstract: The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their ...The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca-activated scramblases, but the mechanisms underlying their Ca-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from , afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.
Validation ReportPDB-ID: 6dz7

SummaryFull reportAbout validation report
DateDeposition: Jul 3, 2018 / Header (metadata) release: Aug 8, 2018 / Map release: Feb 6, 2019 / Last update: Feb 6, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6dz7
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Structure viewerEM map:
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Map

Fileemd_8931.map.gz (map file in CCP4 format, 67109 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
256 pix
1.1 Å/pix.
= 280.602 Å
256 pix
1.1 Å/pix.
= 280.602 Å
256 pix
1.1 Å/pix.
= 280.602 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.0961 Å
Density
Contour Level:0.025 (by author), 0.025 (movie #1):
Minimum - Maximum-0.035463337 - 0.11079124
Average (Standard dev.)0.0006878834 (0.003573915)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions256256256
Origin0.00.00.0
Limit255.0255.0255.0
Spacing256256256
CellA=B=C: 280.6016 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.09610156251.09610156251.0961015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z280.602280.602280.602
α/β/γ90.00090.00090.000
start NX/NY/NZ-16-63-38
NX/NY/NZ727975
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0350.1110.001

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Supplemental data

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Sample components

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Entire afTMEM16 reconstituted in nanodiscs in the absence of Ca2+

EntireName: afTMEM16 reconstituted in nanodiscs in the absence of Ca2+
Number of components: 2

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Component #1: protein, afTMEM16 reconstituted in nanodiscs in the absence of Ca2+

ProteinName: afTMEM16 reconstituted in nanodiscs in the absence of Ca2+
Recombinant expression: No
MassTheoretical: 168 MDa
SourceSpecies: Aspergillus fumigatus (mold)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, Plasma membrane channel protein (Aqy1), putative

ProteinName: Plasma membrane channel protein (Aqy1), putative / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 84.616859 kDa
SourceSpecies: Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) (mold)
Strain: ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 7 mg/ml / pH: 8
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 288 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 62.61 e/Å2 / Illumination mode: OTHER
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 70356
3D reconstructionResolution: 3.89 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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