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- PDB-6qmb: Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in n... -

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Basic information

Entry
Database: PDB / ID: 6qmb
TitleCryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in nanodisc (closed state)
ComponentsPredicted proteinProtein structure prediction
KeywordsMEMBRANE PROTEIN / lipid scrambles / TMEM16
Function / homology: / Alpha-beta plait domain in TMEM16 lipid scramblase / : / Anoctamin / Calcium-activated chloride channel / membrane / identical protein binding / metal ion binding / Plasma membrane channel protein
Function and homology information
Biological speciesNectria haematococca (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKalienkova, V. / Clerico Mosina, V. / Bryner, L. / Oostergetel, G.T. / Dutzler, R. / Paulino, C.
Funding support Switzerland, Netherlands, 2items
OrganizationGrant numberCountry
European Research Council339116, AnoBest. Switzerland
Netherlands Organisation for Scientific Research740.018.016 Netherlands
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.
History
DepositionFeb 1, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 6, 2019Provider: repository / Type: Initial release
Revision 1.1Mar 20, 2019Group: Author supporting evidence / Data collection
Category: em_admin / em_single_particle_entity / pdbx_database_proc
Item: _em_admin.last_update / _em_single_particle_entity.point_symmetry
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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Assembly

Deposited unit
A: Predicted protein
B: Predicted protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,5606
Polymers166,4002
Non-polymers1604
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area8620 Å2
ΔGint-116 kcal/mol
Surface area63810 Å2
MethodPISA

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Components

#1: Protein Predicted protein / Protein structure prediction


Mass: 83200.008 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nectria haematococca (fungus) / Gene: NECHADRAFT_66456 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: C7Z7K1
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: nhTMEM16 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.166 MDa / Experimental value: NO
Source (natural)Organism: Nectria haematococca mpVI 77-13-4 (fungus)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 7.6
Details: 10 mM Hepes 7.6, 150 mM NaCl, 2 mM EGTA. 2.3 mM CaCl2 were added 30 minutes before freezing.
SpecimenConc.: 2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: at 5 mA / Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 49407 X / Calibrated magnification: 49407 X / Nominal defocus max: 3000 nm / Nominal defocus min: 300 nm / Calibrated defocus min: 300 nm / Calibrated defocus max: 3000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 K / Temperature (min): 90 K
Image recordingAverage exposure time: 9 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 9 / Num. of real images: 13844
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 60 / Used frames/image: 1-60

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1RELION2.1bparticle selection
2EPU1.9.1image acquisition
4CTFFIND4.1.8CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION2.1binitial Euler assignment
11RELION3final Euler assignment
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 2440110
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 41631 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingSpace: REAL

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