+Open data
-Basic information
Entry | Database: PDB / ID: 6oy3 | ||||||
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Title | nhTMEM16 L302A +Ca2+ in nanodiscs | ||||||
Components | nhTMEM16 | ||||||
Keywords | LIPID TRANSPORT / TMEM16 / scramblase / anoactamin | ||||||
Function / homology | Function and homology information cortical endoplasmic reticulum / chloride channel activity / identical protein binding / metal ion binding / plasma membrane Similarity search - Function | ||||||
Biological species | Nectria haematococca (fungus) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 Å | ||||||
Authors | Falzone, M. / Lee, B.C. / Accardi, A. | ||||||
Funding support | United States, 1items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6oy3.cif.gz | 217.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6oy3.ent.gz | 173.5 KB | Display | PDB format |
PDBx/mmJSON format | 6oy3.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6oy3_validation.pdf.gz | 879.5 KB | Display | wwPDB validaton report |
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Full document | 6oy3_full_validation.pdf.gz | 892.3 KB | Display | |
Data in XML | 6oy3_validation.xml.gz | 41.1 KB | Display | |
Data in CIF | 6oy3_validation.cif.gz | 62.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/oy/6oy3 ftp://data.pdbj.org/pub/pdb/validation_reports/oy/6oy3 | HTTPS FTP |
-Related structure data
Related structure data | 20221MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | 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 / |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: nhTMEM16 L302A reconstituted in nanodiscs in the presence of Ca2+ Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT |
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Molecular weight | Value: 166 kDa/nm / Experimental value: NO |
Source (natural) | Organism: Nectria haematococca mpVI (fungus) |
Source (recombinant) | Organism: Saccharomyces cerevisiae (brewer's yeast) |
Buffer solution | pH: 8 |
Specimen | Conc.: 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+ |
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 288 K |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER |
Electron lens | Mode: BRIGHT FIELD |
Image recording | Electron dose: 70.7 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 |
-Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||
Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||||||||||
3D reconstruction | Resolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47243 / Symmetry type: POINT |