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- EMDB-4589: Cryo-EM structure of calcium-free nhTMEM16 lipid scramblase in DDM -

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Basic information

Entry
Database: EMDB / ID: EMD-4589
TitleCryo-EM structure of calcium-free nhTMEM16 lipid scramblase in DDM
Map dataNone
Sample
  • Complex: nhTMEM16
    • Protein or peptide: Predicted protein
Keywordsmembrane protein / lipid scrambles / TMEM16
Function / homology
Function and homology information


cortical endoplasmic reticulum / chloride channel activity / metal ion binding / identical protein binding / membrane
Similarity search - Function
: / Alpha-beta plait domain in TMEM16 lipid scramblase / Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Plasma membrane channel protein
Similarity search - Component
Biological speciesNectria haematococca mpVI 77-13-4 (fungus) / Nectria haematococca (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.7 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: 0.033
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.033
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6qm6
  • Surface level: 0.033
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4589.png

Downloads & links

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Map

FileDownload / File: emd_4589.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.01 Å/pix.
x 240 pix.
= 242.88 Å		1.01 Å/pix.
x 240 pix.
= 242.88 Å		1.01 Å/pix.
x 240 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

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.033 / Movie #1: 0.033
Minimum - Maximum-0.092941165 - 0.17777082
Average (Standard dev.)0.000049706632 (±0.0059460257)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 242.87999 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0930.1780.000

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Supplemental data

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Mask #1

Fileemd_4589_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_4589_additional.map
AnnotationRefined not masked nhTMEM16_DDM_noCa EM map where micelle distortion can be observed.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 used during refinement and for...

Fileemd_4589_half_map_1.map
AnnotationHalf map 2 used during refinement and for FSC gold-standard resolution calculation nhTMEM16_DDM_noCa.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_4589_half_map_2.map
AnnotationHalf map 1 used during refinement and for FSC gold-standard resolution calculation nhTMEM16_DDM_noCa.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : nhTMEM16

EntireName: nhTMEM16
Components
  • Complex: nhTMEM16
    • Protein or peptide: Predicted protein

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Supramolecule #1: nhTMEM16

SupramoleculeName: nhTMEM16 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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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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
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 5mA
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: 3351 / 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: 570203
Startup modelType of model: PDB ENTRY
Final reconstructionNumber classes used: 9 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.7 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1b) / Number images used: 238070
Initial angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1b)
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: RELION (ver. 2.1b)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL
Output model

PDB-6qm6:
Cryo-EM structure of calcium-free nhTMEM16 lipid scramblase in DDM

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