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

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Database: PDB / ID: 6qmb
TitleCryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in nanodisc (closed state)
ComponentsPredicted proteinPrediction
KeywordsMEMBRANE PROTEIN / membrane protein / lipid scrambles / TMEM16
Function / homologyAnoctamin / Calcium-activated chloride channel / integral component of membrane / identical protein binding / metal ion binding / Uncharacterized protein
Function and homology information
Specimen sourceNectria haematococca (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.6 Å resolution
AuthorsKalienkova, V. / Clerico Mosina, V. / Bryner, L. / Oostergetel, G.T. / 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 Report
SummaryFull reportAbout validation report
DateDeposition: Feb 1, 2019 / Release: Mar 6, 2019
RevisionDateData content typeGroupCategoryItemProviderType
1.0Mar 6, 2019Structure modelrepositoryInitial release
1.1Mar 20, 2019Structure modelAuthor supporting evidence / Data collectionem_admin / em_single_particle_entity / pdbx_database_proc_em_admin.last_update / _em_single_particle_entity.point_symmetry

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

Theoretical massNumber of molelcules
Total (without water)166,5606

TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area (Å2)8620
ΔGint (kcal/M)-116
Surface area (Å2)63810


#1: Protein/peptide Predicted protein / Prediction

Mass: 83200.008 Da / Num. of mol.: 2 / Source: (gene. exp.) Nectria haematococca (fungus) / Gene: NECHADRAFT_66456 / Production host: Saccharomyces cerevisiae (baker's yeast) / References: UniProt: C7Z7K1
#2: Chemical

Mass: 40.078 Da / Num. of mol.: 4 / Formula: Ca / Calcium

Experimental details


EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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 (baker's yeast)
Buffer solutionDetails: 10 mM Hepes 7.6, 150 mM NaCl, 2 mM EGTA. 2.3 mM CaCl2 were added 30 minutes before freezing.
pH: 7.6
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 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 kelvins

Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyMicroscope model: 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 / Calibrated magnification: 49407 / 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 microns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 105 kelvins / Temperature (min): 90 kelvins
Image recordingAverage exposure time: 9 sec. / Electron dose: 52 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Number of grids imaged: 9 / Number 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


SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
1RELION2.1bparticle selection
2EPU1.9.1image acquisition
4CTFFIND4.1.8CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
10RELION2.1binitial Euler assignment
11RELION3.0final Euler assignment
13RELION3.03D reconstruction
Particle selectionNumber of particles selected: 2440110
SymmetryPoint symmetry: C2
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 41631 / Algorithm: BACK PROJECTION / Symmetry type: POINT
Atomic model buildingRef space: REAL

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