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- PDB-8tps: nhTMEM16 A444P mutant in lipid nanodiscs with MSP1E3 scaffold pro... -

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Basic information

Entry
Database: PDB / ID: 8tps
TitlenhTMEM16 A444P mutant in lipid nanodiscs with MSP1E3 scaffold protein in the presence of Ca2+ (bent TM6)
ComponentsLipid scramblase nhTMEM16
KeywordsLIPID TRANSPORT / membrane protein / lipid scramblase / TMEM16
Function / homology
Function and homology information


identical protein binding / membrane / metal ion binding
Similarity search - Function
: / Alpha-beta plait domain in TMEM16 lipid scramblase / Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Chem-PGW / Plasma membrane channel protein
Similarity search - Component
Biological speciesFusarium vanettenii 77-13-4 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 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 4, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 8, 2024Provider: repository / Type: Initial release
Revision 1.1Oct 30, 2024Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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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)169,7168
Polymers166,6402
Non-polymers3,0766
Water00
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: 83320.094 Da / Num. of mol.: 2 / Mutation: A444P
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.: 2 / Source method: obtained synthetically / Formula: Ca / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-PGW / (1R)-2-{[(S)-{[(2S)-2,3-dihydroxypropyl]oxy}(hydroxy)phosphoryl]oxy}-1-[(hexadecanoyloxy)methyl]ethyl (9Z)-octadec-9-enoate / 1-Palmitoyl-2-Oleoyl-sn-Glycero-3-[Phospho-(1-glycerol)] / PHOSPHATIDYLGLYCEROL


Mass: 749.007 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C40H77O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
Has ligand of interestY
Has protein modificationN

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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
Molecular weightExperimental value: NO
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

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 1900 nm / Nominal defocus min: 900 nm
Image recordingAverage exposure time: 2 sec. / Electron dose: 59.2 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 11976
EM imaging opticsEnergyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategory
1RELION3.1.2particle selection
2Leginonimage acquisition
4RELION3.1.2CTF correction
7Coot0.8.9model fitting
9PHENIX1.20.1model refinement
10RELION3.1.2initial Euler assignment
11RELION3.1.2final Euler assignment
12RELION3.1.2classification
13RELION3.1.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4885672
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17404 / Symmetry type: POINT
Atomic model buildingPDB-ID: 6QM9

6qm9


Accession code: 6QM9 / Source name: PDB / Type: experimental model

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