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- PDB-8tok: nhTMEM16 lipid scramblase in lipid nanodiscs with MSP2N2 scaffold... -

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Basic information

Entry
Database: PDB / ID: 8tok
TitlenhTMEM16 lipid scramblase in lipid nanodiscs with MSP2N2 scaffold protein in the presence of Ca2+ (open state)
ComponentsLipid scramblase nhTMEM16
KeywordsLIPID TRANSPORT / membrane protein / lipid scramblase / TMEM16
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 speciesFusarium vanettenii 77-13-4 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.84 Å
AuthorsFeng, Z. / Accardi, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM106717 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis of closed groove scrambling by a TMEM16 protein.
Authors: Zhang Feng / Omar E Alvarenga / Alessio Accardi /
Abstract: Activation of Ca-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by ...Activation of Ca-dependent TMEM16 scramblases induces phosphatidylserine externalization, a key step in multiple signaling processes. Current models suggest that the TMEM16s scramble lipids by deforming the membrane near a hydrophilic groove and that Ca dependence arises from the different association of lipids with an open or closed groove. However, the molecular rearrangements underlying groove opening and how lipids reorganize outside the closed groove remain unknown. Here we directly visualize how lipids associate at the closed groove of Ca-bound fungal nhTMEM16 in nanodiscs using cryo-EM. Functional experiments pinpoint lipid-protein interaction sites critical for closed groove scrambling. Structural and functional analyses suggest groove opening entails the sequential appearance of two π-helical turns in the groove-lining TM6 helix and identify critical rearrangements. Finally, we show that the choice of scaffold protein and lipids affects the conformations of nhTMEM16 and their distribution, highlighting a key role of these factors in cryo-EM structure determination.
History
DepositionAug 3, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Lipid scramblase nhTMEM16
B: Lipid scramblase nhTMEM16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,7486
Polymers166,5882
Non-polymers1604
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Lipid scramblase nhTMEM16


Mass: 83294.062 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Fusarium vanettenii 77-13-4 (fungus) / Gene: NECHADRAFT_66456 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: C7Z7K1
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Dimeric lipid scramblase nhTMEM16 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Fusarium vanettenii 77-13-4 (fungus)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris1
2150 mMKCl1
30.5 mMCaCl21
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 3.4 sec. / Electron dose: 30 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 9493
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1RELION4.0.0particle selection
2EPUimage acquisition
4RELION4.0.0CTF correction
7Coot0.8.9model fitting
9PHENIX1.20.1model refinement
10RELION4.0.0initial Euler assignment
11RELION4.0.0final Euler assignment
12RELION4.0.0classification
13RELION4.0.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1948732
3D reconstructionResolution: 3.84 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 250756 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6QMA

6qma


Accession code: 6QMA / Source name: PDB / Type: experimental model

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