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- EMDB-20221: nhTMEM16 L302A +Ca2+ in nanodiscs -

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Basic information

Entry
Database: EMDB / ID: EMD-20221
TitlenhTMEM16 L302A +Ca2+ in nanodiscs
Map dataFinal EM map for nhTMEM16 L302A/nanodisc complex with C2 symmetry. Used for model buidling.
Sample
  • Complex: nhTMEM16 L302A reconstituted in nanodiscs in the presence of Ca2+
    • Protein or peptide: nhTMEM16
  • Ligand: CALCIUM IONCalcium
KeywordsTMEM16 / scramblase / lipid transport / anoactamin
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 mpVI (fungus) / Nectria haematococca (strain 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI) (fungus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.0 Å
AuthorsFalzone M / Lee BC
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)5R01GM106717 United States
CitationJournal: Nat Commun / Year: 2019
Title: Dynamic modulation of the lipid translocation groove generates a conductive ion channel in Ca-bound nhTMEM16.
Authors: George Khelashvili / Maria E Falzone / Xiaolu Cheng / Byoung-Cheol Lee / Alessio Accardi / Harel Weinstein /
Abstract: Both lipid and ion translocation by Ca-regulated TMEM16 transmembrane proteins utilizes a membrane-exposed hydrophilic groove. Several conformations of the groove are observed in TMEM16 protein ...Both lipid and ion translocation by Ca-regulated TMEM16 transmembrane proteins utilizes a membrane-exposed hydrophilic groove. Several conformations of the groove are observed in TMEM16 protein structures, but how these conformations form, and what functions they support, remains unknown. From analyses of atomistic molecular dynamics simulations of Ca-bound nhTMEM16 we find that the mechanism of a conformational transition of the groove from membrane-exposed to occluded from the membrane involves the repositioning of transmembrane helix 4 (TM4) following its disengagement from a TM3/TM4 interaction interface. Residue L302 is a key element in the hydrophobic TM3/TM4 interaction patch that braces the open-groove conformation, which should be changed by an L302A mutation. The structure of the L302A mutant determined by cryogenic electron microscopy (cryo-EM) reveals a partially closed groove that could translocate ions, but not lipids. This is corroborated with functional assays showing severely impaired lipid scrambling, but robust channel activity by L302A.
History
DepositionMay 14, 2019-
Header (metadata) releaseOct 2, 2019-
Map releaseNov 6, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0236
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.0236
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6oy3
  • Surface level: 0.0236
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20221.png

Downloads & links

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Map

FileDownload / File: emd_20221.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFinal EM map for nhTMEM16 L302A/nanodisc complex with C2 symmetry. Used for model buidling.
Voxel sizeX=Y=Z: 1.0961 Å
Density
Contour LevelBy AUTHOR: 0.0236 / Movie #1: 0.0236
Minimum - Maximum-0.13025297 - 0.17961413
Average (Standard dev.)0.000010987753 (±0.004067377)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 280.6016 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.09610156251.09610156251.0961015625
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z280.602280.602280.602
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1300.1800.000

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Supplemental data

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Additional map: Final EM map for nhTMEM16 L302A/nanodisc complex with...

Fileemd_20221_additional_1.map
AnnotationFinal EM map for nhTMEM16 L302A/nanodisc complex with C1 symmetry. Used to analyze effects of nhTMEM16
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unfiltered map from refinement of the nhTMEM16 L302A/nanodisc....

Fileemd_20221_additional_2.map
AnnotationUnfiltered map from refinement of the nhTMEM16 L302A/nanodisc. Used to assist model building.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : nhTMEM16 L302A reconstituted in nanodiscs in the presence of Ca2+

EntireName: nhTMEM16 L302A reconstituted in nanodiscs in the presence of Ca2+
Components
  • Complex: nhTMEM16 L302A reconstituted in nanodiscs in the presence of Ca2+
    • Protein or peptide: nhTMEM16
  • Ligand: CALCIUM IONCalcium

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Supramolecule #1: nhTMEM16 L302A reconstituted in nanodiscs in the presence of Ca2+

SupramoleculeName: nhTMEM16 L302A reconstituted in nanodiscs in the presence of Ca2+
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Nectria haematococca mpVI (fungus)
Molecular weightTheoretical: 166 kDa/nm

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Macromolecule #1: nhTMEM16

MacromoleculeName: nhTMEM16 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Nectria haematococca (strain 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI) (fungus)
Strain: 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI
Molecular weightTheoretical: 83.15793 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 CVVAAGALIV 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

Concentration6.5 mg/mL
BufferpH: 8
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 288 K / Instrument: FEI VITROBOT MARK IV
DetailsnhTMEM16 L302A reconstituted in nanodiscs in the absence of Ca2+

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Average electron dose: 70.7 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

4wis

Initial angle assignmentType: OTHER
Final angle assignmentType: ANGULAR RECONSTITUTION
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 47243
FSC plot (resolution estimation)

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