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- EMDB-4594: Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in n... -
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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-4594 | |||||||||
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Title | Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in nanodisc (closed state) | |||||||||
![]() | None | |||||||||
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![]() | membrane protein / lipid scrambles / TMEM16 | |||||||||
Function / homology | ![]() cortical endoplasmic reticulum / chloride channel activity / identical protein binding / metal ion binding / plasma membrane Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.6 Å | |||||||||
![]() | Kalienkova V / Clerico Mosina V | |||||||||
Funding support | ![]() ![]()
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![]() | ![]() 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 |
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Structure visualization
Movie |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 48.7 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 18.8 KB 18.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 8.6 KB | Display | ![]() |
Images | ![]() | 144.8 KB | ||
Masks | ![]() | 52.7 MB | ![]() | |
Filedesc metadata | ![]() | 6.5 KB | ||
Others | ![]() ![]() | 39.6 MB 39.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6qmbMC ![]() 4587C ![]() 4588C ![]() 4589C ![]() 4592C ![]() 4593C ![]() 6qm4C ![]() 6qm5C ![]() 6qm6C ![]() 6qm9C ![]() 6qmaC M: atomic model generated by this map C: citing same article ( |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | None | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.012 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
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Projections & Slices |
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Density Histograms |
-Half map: Half map 1 used during refinement and for...
File | emd_4594_half_map_1.map | ||||||||||||
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Annotation | Half map 1 used during refinement and for FSC gold-standard resolution calculation nhTMEM16_2N2_Ca. | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2 used during refinement and for...
File | emd_4594_half_map_2.map | ||||||||||||
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Annotation | Half map 2 used during refinement and for FSC gold-standard resolution calculation nhTMEM16_2N2_Ca. | ||||||||||||
Projections & Slices |
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Density Histograms |
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Sample components
-Entire : nhTMEM16
Entire | Name: nhTMEM16 |
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Components |
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-Supramolecule #1: nhTMEM16
Supramolecule | Name: nhTMEM16 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 166 KDa |
-Macromolecule #1: Predicted protein
Macromolecule | Name: Predicted protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 83.200008 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: MSNLKDFSQP GSGQESNFGV DFVIHYKVPA AERDEAEAGF VQLIRALTTV GLATEVRHGE NESLLVFVKV ASPDLFAKQV YRARLGDWL HGVRVSAPHN DIAQALQDEP VVEAERLRLI YLMITKPHNE GGAGVTPTNA KWKHVESIFP LHSHSFNKEW I KKWSSKYT ...String: MSNLKDFSQP GSGQESNFGV DFVIHYKVPA AERDEAEAGF VQLIRALTTV GLATEVRHGE NESLLVFVKV ASPDLFAKQV YRARLGDWL HGVRVSAPHN DIAQALQDEP VVEAERLRLI YLMITKPHNE GGAGVTPTNA KWKHVESIFP LHSHSFNKEW I KKWSSKYT LEQTDIDNIR DKFGESVAFY FAFLRSYFRF LVIPSAFGFG AWLLLGQFSY LYALLCGLWS VVFFEYWKKQ EV DLAVQWG VRGVSSIQQS RPEFEWEHEA EDPITGEPVK VYPPMKRVKT QLLQIPFALA CVVALGALIV TCNSLEVFIN EVY SGPGKQ YLGFLPTIFL VIGTPTISGV LMGAAEKLNA MENYATVDAH DAALIQKQFV LNFMTSYMAL FFTAFVYIPF GHIL HPFLN FWRATAQTLT FSEKELPTRE FQINPARISN QMFYFTVTAQ IVNFATEVVV PYIKQQAFQK AKQLKSGSKV QEDHE EEAE FLQRVREECT LEEYDVSGDY REMVMQFGYV AMFSVAWPLA ACCFLVNNWV ELRSDALKIA ISSRRPIPWR TDSIGP WLT ALSFLSWLGS ITSSAIVYLC SNSKNGTQGE ASPLKAWGLL LSILFAEHFY LVVQLAVRFV LSKLDSPGLQ KERKERF QT KKRLLQENLG QDAAEEAAAP GIEHSEKITR EALEEEARQA SIRGHGTPEE MFWQRQRGMQ ETIEIGRRMI EQQLAAGK N GKKSAPAVPS EKAS UniProtKB: Plasma membrane channel protein |
-Macromolecule #2: CALCIUM ION
Macromolecule | Name: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: CA |
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Molecular weight | Theoretical: 40.078 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Concentration | 2 mg/mL |
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Buffer | pH: 7.6 Details: 10 mM Hepes 7.6, 150 mM NaCl, 2 mM EGTA. 2.3 mM CaCl2 were added 30 minutes before freezing. |
Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: at 5 mA |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Temperature | Min: 90.0 K / Max: 105.0 K |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-60 / Number grids imaged: 9 / Number real images: 13844 / Average exposure time: 9.0 sec. / Average electron dose: 52.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 100.0 µm / Calibrated defocus max: 3.0 µm / Calibrated defocus min: 0.3 µm / Calibrated magnification: 49407 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 0.3 µm / Nominal magnification: 49407 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Refinement | Space: REAL |
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Output model | ![]() PDB-6qmb: |