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- EMDB-4588: Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in DDM -

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Basic information

Entry
Database: EMDB / ID: EMD-4588
TitleCryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in DDM
Map dataNone
Sample
  • Complex: nhTMEM16
    • Protein or peptide: Predicted protein
  • Ligand: CALCIUM ION
Keywordsmembrane protein / lipid scrambles / TMEM16
Function / homology: / Alpha-beta plait domain in TMEM16 lipid scramblase / Anoctamin / : / Calcium-activated chloride channel / identical protein binding / membrane / metal ion binding / Plasma membrane channel protein
Function and homology information
Biological speciesNectria haematococca mpVI 77-13-4 (fungus) / Nectria haematococca (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsKalienkova V / Clerico Mosina V
Funding support Switzerland, Netherlands, 2 items
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, 2019-
Header (metadata) releaseMar 6, 2019-
Map releaseMar 6, 2019-
UpdateMay 15, 2024-
Current statusMay 15, 2024Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0431
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0431
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qm5
  • Surface level: 0.0431
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4588.png

Downloads & links

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Map

FileDownload / File: emd_4588.map.gz / Format: CCP4 / Size: 40.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 220 pix.
= 222.64 Å		1.01 Å/pix.
x 220 pix.
= 222.64 Å		1.01 Å/pix.
x 220 pix.
= 222.64 Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.012 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0431 / Movie #1: 0.0431
Minimum - Maximum-0.17526333 - 0.23554112
Average (Standard dev.)0.00007685376 (±0.008181418)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions220220220
Spacing220220220
CellA=B=C: 222.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0121.0121.012
M x/y/z220220220
origin x/y/z0.0000.0000.000
length x/y/z222.640222.640222.640
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS220220220
D min/max/mean-0.1750.2360.000

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Supplemental data

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Mask #1

Fileemd_4588_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Refined not masked nhTMEM16 DDM Ca EM map where micelle...

Fileemd_4588_additional.map
AnnotationRefined not masked nhTMEM16_DDM_Ca EM map where micelle distortion can be observed.
Projections & Slices
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Half map: Half map 2 used during refinement and for...

Fileemd_4588_half_map_1.map
AnnotationHalf map 2 used during refinement and for FSC gold-standard resolution calculation nhTMEM16_DDM_Ca.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 used during refinement and for...

Fileemd_4588_half_map_2.map
AnnotationHalf map 1 used during refinement and for FSC gold-standard resolution calculation nhTMEM16_DDM_Ca.
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Sample components

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Entire : nhTMEM16

EntireName: nhTMEM16
Components
  • Complex: nhTMEM16
    • Protein or peptide: Predicted protein
  • Ligand: CALCIUM ION

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Supramolecule #1: nhTMEM16

SupramoleculeName: nhTMEM16 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Nectria haematococca mpVI 77-13-4 (fungus)
Molecular weightTheoretical: 166 KDa

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Macromolecule #1: Predicted protein

MacromoleculeName: Predicted protein / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Nectria haematococca (fungus)
Molecular weightTheoretical: 83.200008 KDa
Recombinant expressionOrganism: Saccharomyces cerevisiae (brewer's yeast)
SequenceString: 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

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Macromolecule #2: CALCIUM ION

MacromoleculeName: CALCIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: CA
Molecular weightTheoretical: 40.078 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration3.3 mg/mL
BufferpH: 7.6
Details: 5 mM Hepes 7.6, 150 mM NaCl, 0.03% DDM, 2 mM EGTA. 2.3 mM CaCl2 were added 30 minutes before freezing.
GridModel: 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
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
TemperatureMin: 90.0 K / Max: 105.0 K
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm 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: 2521 / Average exposure time: 9.0 sec. / Average electron dose: 52.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 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 stageSpecimen 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

Particle selectionNumber selected: 251693
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4wis

Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 120086
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1b)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 3.0)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

4wis


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL
Output model

PDB-6qm5:
Cryo-EM structure of calcium-bound nhTMEM16 lipid scramblase in DDM

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