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- PDB-7rxh: afTMEM16 in C18 lipid nanodiscs with MSP1E3 scaffold protein in t... -

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Basic information

Entry
Database: PDB / ID: 7rxh
TitleafTMEM16 in C18 lipid nanodiscs with MSP1E3 scaffold protein in the presence of Ca2+, monomer with extra lipids
ComponentsafTMEM16 lipid scramblase
KeywordsLIPID TRANSPORT / TMEM16 / lipid scrambling
Function / homology
Function and homology information


phospholipid scramblase activity / cortical endoplasmic reticulum / phospholipid translocation / chloride channel activity / voltage-gated calcium channel activity / chloride transmembrane transport / monoatomic ion transmembrane transport / membrane
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 (Aqy1), putative
Similarity search - Component
Biological speciesNeosartorya fumigata (mold)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsFalzone, M.E. / 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 Commun / Year: 2022
Title: TMEM16 scramblases thin the membrane to enable lipid scrambling.
Authors: Maria E Falzone / Zhang Feng / Omar E Alvarenga / Yangang Pan / ByoungCheol Lee / Xiaolu Cheng / Eva Fortea / Simon Scheuring / Alessio Accardi /
Abstract: TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. Scrambling was proposed to occur with lipid headgroups moving between leaflets ...TMEM16 scramblases dissipate the plasma membrane lipid asymmetry to activate multiple eukaryotic cellular pathways. Scrambling was proposed to occur with lipid headgroups moving between leaflets through a membrane-spanning hydrophilic groove. Direct information on lipid-groove interactions is lacking. We report the 2.3 Å resolution cryogenic electron microscopy structure of the nanodisc-reconstituted Ca-bound afTMEM16 scramblase showing how rearrangement of individual lipids at the open pathway results in pronounced membrane thinning. Only the groove's intracellular vestibule contacts lipids, and mutagenesis suggests scrambling does not require specific protein-lipid interactions with the extracellular vestibule. We find scrambling can occur outside a closed groove in thinner membranes and is inhibited in thicker membranes, despite an open pathway. Our results show afTMEM16 thins the membrane to enable scrambling and that an open hydrophilic pathway is not a structural requirement to allow rapid transbilayer movement of lipids. This mechanism could be extended to other scramblases lacking a hydrophilic groove.
History
DepositionAug 23, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0May 18, 2022Provider: repository / Type: Initial release
Revision 1.1May 25, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jun 5, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: afTMEM16 lipid scramblase
hetero molecules


Theoretical massNumber of molelcules
Total (without water)96,68119
Polymers84,6171
Non-polymers12,06418
Water43224
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein afTMEM16 lipid scramblase


Mass: 84616.859 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neosartorya fumigata (strain CEA10 / CBS 144.89 / FGSC A1163) (mold)
Strain: CEA10 / CBS 144.89 / FGSC A1163 / Gene: AFUA_4G02970 / Production host: Saccharomyces cerevisiae (brewer's yeast) / References: UniProt: Q4WA18
#2: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#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.: 16 / Source method: obtained synthetically / Formula: C40H77O10P / Feature type: SUBJECT OF INVESTIGATION / Comment: phospholipid*YM
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 24 / Source method: isolated from a natural source / Formula: H2O
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: afTMEM16 scramblase C18 lipid nanodiscs with MSP1E3 scaffold in the presence of Ca2+, monomer with additional lipids
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Aspergillus fumigatus (mold)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 %

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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
Image recordingElectron dose: 42.8227 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassificationNB
PHENIXrefinement
PDB_EXTRACT3.27data extraction
EM softwareName: SerialEM / Category: image acquisition
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 1058829
Details: Sorted particles for the C2 dimer reconstruction were symmetry expanded and further classified with a mask on one monomer with and without local alignment with varying T values. These ...Details: Sorted particles for the C2 dimer reconstruction were symmetry expanded and further classified with a mask on one monomer with and without local alignment with varying T values. These 1058829 sorted into a class with strong density for additional lipids.
Symmetry type: POINT
RefinementCross valid method: THROUGHOUT
Displacement parametersBiso max: 93.46 Å2 / Biso mean: 35.5277 Å2 / Biso min: 12.35 Å2

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