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- PDB-6e0h: PDB: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+ -

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Basic information

Entry
Database: PDB / ID: 6e0h
TitlePDB: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+
ComponentsPlasma membrane channel protein (Aqy1), putative
KeywordsLIPID TRANSPORT / scramblase / Ca2+-activated / membrane-reorganization
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
Plasma membrane channel protein (Aqy1), putative
Similarity search - Component
Biological speciesNeosartorya fumigata (mold)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.05 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)5R01GM106717 United States
CitationJournal: Elife / Year: 2019
Title: Structural basis of Ca-dependent activation and lipid transport by a TMEM16 scramblase.
Authors: Maria E Falzone / Jan Rheinberger / Byoung-Cheol Lee / Thasin Peyear / Linda Sasset / Ashleigh M Raczkowski / Edward T Eng / Annarita Di Lorenzo / Olaf S Andersen / Crina M Nimigean / Alessio Accardi /
Abstract: The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their ...The lipid distribution of plasma membranes of eukaryotic cells is asymmetric and phospholipid scramblases disrupt this asymmetry by mediating the rapid, nonselective transport of lipids down their concentration gradients. As a result, phosphatidylserine is exposed to the outer leaflet of membrane, an important step in extracellular signaling networks controlling processes such as apoptosis, blood coagulation, membrane fusion and repair. Several TMEM16 family members have been identified as Ca-activated scramblases, but the mechanisms underlying their Ca-dependent gating and their effects on the surrounding lipid bilayer remain poorly understood. Here, we describe three high-resolution cryo-electron microscopy structures of a fungal scramblase from , afTMEM16, reconstituted in lipid nanodiscs. These structures reveal that Ca-dependent activation of the scramblase entails global rearrangement of the transmembrane and cytosolic domains. These structures, together with functional experiments, suggest that activation of the protein thins the membrane near the transport pathway to facilitate rapid transbilayer lipid movement.
History
DepositionJul 6, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 6, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Other / Category: atom_sites / cell / pdbx_audit_support
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB / _pdbx_audit_support.funding_organization
Revision 1.2Mar 13, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_conn_angle / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_conn_angle.ptnr1_auth_comp_id / _pdbx_struct_conn_angle.ptnr1_auth_seq_id / _pdbx_struct_conn_angle.ptnr1_label_atom_id / _pdbx_struct_conn_angle.ptnr1_label_comp_id / _pdbx_struct_conn_angle.ptnr1_label_seq_id / _pdbx_struct_conn_angle.ptnr2_auth_seq_id / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_struct_conn_angle.ptnr3_auth_comp_id / _pdbx_struct_conn_angle.ptnr3_auth_seq_id / _pdbx_struct_conn_angle.ptnr3_label_atom_id / _pdbx_struct_conn_angle.ptnr3_label_comp_id / _pdbx_struct_conn_angle.ptnr3_label_seq_id / _pdbx_struct_conn_angle.value / _struct_conn.pdbx_dist_value / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

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Assembly

Deposited unit
B: Plasma membrane channel protein (Aqy1), putative
A: Plasma membrane channel protein (Aqy1), putative
hetero molecules


Theoretical massNumber of molelcules
Total (without water)169,3946
Polymers169,2342
Non-polymers1604
Water00
1


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

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Components

#1: Protein Plasma membrane channel protein (Aqy1), putative


Mass: 84616.859 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Neosartorya fumigata (strain ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100) (mold)
Strain: ATCC MYA-4609 / Af293 / CBS 101355 / FGSC A1100 / 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.: 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: afTMEM16 reconstituted in nanodiscs in the presence of Ca2+
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 168 kDa/nm / Experimental value: NO
Source (natural)Organism: Aspergillus fumigatus (mold)
Source (recombinant)Organism: Saccharomyces cerevisiae (brewer's yeast)
Buffer solutionpH: 8
SpecimenConc.: 7 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: afTMEM16 reconstituted in nanodiscs in the presence 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 FIELD
Image recordingElectron dose: 69.97 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

Software
NameVersionClassification
phenix.real_space_refine1.13_2998refinement
PHENIX1.13_2998refinement
EM software
IDNameVersionCategory
2Leginonimage acquisition
4CTFFIND4CTF correction
9PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 4.05 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37146 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library
Refinement stepCycle: 1 /
ProteinNucleic acidLigandSolvent
Num. atoms9520 0 4 0
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00889770
ELECTRON MICROSCOPYf_angle_d1.087913276
ELECTRON MICROSCOPYf_chiral_restr0.0611504
ELECTRON MICROSCOPYf_plane_restr0.00791640
ELECTRON MICROSCOPYf_dihedral_angle_d13.58765740

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