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- EMDB-4592: Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in n... -

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Basic information

Entry
Database: EMDB / ID: 4592
TitleCryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in nanodisc (open state)
Map data
SamplenhTMEM16:
Predicted proteinPrediction / ligand
Function / homologyAnoctamin / Calcium-activated chloride channel / integral component of membrane / identical protein binding / metal ion binding / Uncharacterized protein
Function and homology information
SourceNectria haematococca mpVI 77-13-4 (fungus) / Nectria haematococca (fungus)
Methodsingle particle reconstruction / cryo EM / 3.6 Å resolution
AuthorsKalienkova V / Clerico Mosina V / Bryner L / Oostergetel GT / Dutzler R / Paulino C
CitationJournal: Elife / Year: 2019
Title: Stepwise activation mechanism of the scramblase nhTMEM16 revealed by cryo-EM.
Authors: Valeria Kalienkova / Vanessa Clerico Mosina / Laura Bryner / Gert T Oostergetel / Raimund Dutzler / Cristina Paulino
Abstract: Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca-dependent lipid ...Scramblases catalyze the movement of lipids between both leaflets of a bilayer. Whereas the X-ray structure of the protein nhTMEM16 has previously revealed the architecture of a Ca-dependent lipid scramblase, its regulation mechanism has remained elusive. Here, we have used cryo-electron microscopy and functional assays to address this question. Ca-bound and Ca-free conformations of nhTMEM16 in detergent and lipid nanodiscs illustrate the interactions with its environment and they reveal the conformational changes underlying its activation. In this process, Ca binding induces a stepwise transition of the catalytic subunit cavity, converting a closed cavity that is shielded from the membrane in the absence of ligand, into a polar furrow that becomes accessible to lipid headgroups in the Ca-bound state. Additionally, our structures demonstrate how nhTMEM16 distorts the membrane at both entrances of the subunit cavity, thereby decreasing the energy barrier for lipid movement.
Validation ReportPDB-ID: 6qm9

SummaryFull reportAbout validation report
DateDeposition: Feb 1, 2019 / Header (metadata) release: Mar 6, 2019 / Map release: Mar 6, 2019 / Last update: Mar 20, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.065
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6qm9
  • Surface level: 0.065
  • 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_4592.map.gz (map file in CCP4 format, 55297 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
240 pix
1.01 Å/pix.
= 242.88 Å
240 pix
1.01 Å/pix.
= 242.88 Å
240 pix
1.01 Å/pix.
= 242.88 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:0.03 (by author), 0.065 (movie #1):
Minimum - Maximum0.0 - 0.66604334
Average (Standard dev.)0.018058486 (0.098875)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions240240240
Origin0.00.00.0
Limit239.0239.0239.0
Spacing240240240
CellA=B=C: 242.87999 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/z240240240
origin x/y/z0.0000.0000.000
length x/y/z242.880242.880242.880
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.1950.3310.001

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Supplemental data

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Mask #1

Fileemd_4592_msk_1.map
Projections & Slices
AxesZYX
Projections
Slices (1/2)
Density Histograms

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Sample components

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Entire nhTMEM16

EntireName: nhTMEM16 / Number of components: 3

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Component #1: protein, nhTMEM16

ProteinName: nhTMEM16 / Recombinant expression: No
MassTheoretical: 166 kDa
SourceSpecies: Nectria haematococca mpVI 77-13-4 (fungus)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #2: protein, Predicted protein

ProteinName: Predicted proteinPrediction / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 83.200008 kDa
SourceSpecies: Nectria haematococca (fungus)
Source (engineered)Expression System: Saccharomyces cerevisiae (baker's yeast)

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Component #3: ligand, CALCIUM ION

LigandName: CALCIUM IONCalcium / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 4.007805 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionSpecimen conc.: 2 mg/ml
Buffer solution: 10 mM Hepes 7.6, 150 mM NaCl, 2 mM EGTA. 2.3 mM CaCl2 were added 30 minutes before freezing.
pH: 7.6
Support filmat 5mA
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: 13844

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 71175
3D reconstructionAlgorithm: BACK PROJECTION / Software: RELION / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Modeling #1Refinement space: REAL
Output model

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