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- PDB-6oy3: nhTMEM16 L302A +Ca2+ in nanodiscs -

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Basic information

Entry
Database: PDB / ID: 6oy3
TitlenhTMEM16 L302A +Ca2+ in nanodiscs
ComponentsnhTMEM16
KeywordsLIPID TRANSPORT / TMEM16 / scramblase / 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 (fungus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å
AuthorsFalzone, M. / Lee, B.C. / Accardi, A.
Funding support United States, 1items
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, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 6, 2019Provider: repository / Type: Initial release
Revision 1.1Jan 8, 2020Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization
Revision 1.2Nov 18, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year
Revision 1.3Mar 20, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
B: nhTMEM16
A: nhTMEM16
hetero molecules


Theoretical massNumber of molelcules
Total (without water)166,4766
Polymers166,3162
Non-polymers1604
Water0
1


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

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Components

#1: Protein nhTMEM16


Mass: 83157.930 Da / Num. of mol.: 2 / Mutation: L302A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Nectria haematococca (strain 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI) (fungus)
Strain: 77-13-4 / ATCC MYA-4622 / FGSC 9596 / MPVI / 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

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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: nhTMEM16 L302A reconstituted in nanodiscs in the presence of Ca2+
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 166 kDa/nm / Experimental value: NO
Source (natural)Organism: Nectria haematococca mpVI (fungus)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
SpecimenConc.: 6.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: nhTMEM16 L302A reconstituted in nanodiscs in the absence of Ca2+
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: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 70.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

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Processing

EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
9PHENIXmodel refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47243 / Symmetry type: POINT

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