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- PDB-6r65: Crystal Structure of human TMEM16K / Anoctamin 10 (Form 2) -

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Basic information

Entry
Database: PDB / ID: 6r65
TitleCrystal Structure of human TMEM16K / Anoctamin 10 (Form 2)
ComponentsAnoctamin-10Calcium-dependent chloride channel
KeywordsLIPID TRANSPORT / MEMBRANE PROTEIN / CALCIUM ACTIVATION / TRANSPORT PROTEIN / Structural Genomics / Structural Genomics Consortium / SGC
Function / homology
Function and homology information


intracellularly calcium-gated chloride channel activity / calcium-activated cation channel activity / chloride transport / chloride channel activity / monoatomic cation transport / chloride transmembrane transport / monoatomic ion transmembrane transport / Stimuli-sensing channels / Induction of Cell-Cell Fusion / intracellular membrane-bounded organelle ...intracellularly calcium-gated chloride channel activity / calcium-activated cation channel activity / chloride transport / chloride channel activity / monoatomic cation transport / chloride transmembrane transport / monoatomic ion transmembrane transport / Stimuli-sensing channels / Induction of Cell-Cell Fusion / intracellular membrane-bounded organelle / membrane / plasma membrane
Similarity search - Function
Anoctamin / : / Calcium-activated chloride channel
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.5 Å
AuthorsBushell, S.R. / Pike, A.C.W. / Chu, A. / Tessitore, A. / Rotty, B. / Mukhopadhyay, S. / Kupinska, K. / Shrestha, L. / Borkowska, O. / Chalk, R. ...Bushell, S.R. / Pike, A.C.W. / Chu, A. / Tessitore, A. / Rotty, B. / Mukhopadhyay, S. / Kupinska, K. / Shrestha, L. / Borkowska, O. / Chalk, R. / Burgess-Brown, N.A. / Love, J. / Edwards, A.M. / Arrowsmith, C.H. / Bountra, C. / Carpenter, E.P. / Structural Genomics Consortium (SGC)
Funding support United Kingdom, 2items
OrganizationGrant numberCountry
Wellcome Trust106169/Z/14/Z United Kingdom
European Commission115766 United Kingdom
CitationJournal: Nat Commun / Year: 2019
Title: The structural basis of lipid scrambling and inactivation in the endoplasmic reticulum scramblase TMEM16K.
Authors: Simon R Bushell / Ashley C W Pike / Maria E Falzone / Nils J G Rorsman / Chau M Ta / Robin A Corey / Thomas D Newport / John C Christianson / Lara F Scofano / Chitra A Shintre / Annamaria ...Authors: Simon R Bushell / Ashley C W Pike / Maria E Falzone / Nils J G Rorsman / Chau M Ta / Robin A Corey / Thomas D Newport / John C Christianson / Lara F Scofano / Chitra A Shintre / Annamaria Tessitore / Amy Chu / Qinrui Wang / Leela Shrestha / Shubhashish M M Mukhopadhyay / James D Love / Nicola A Burgess-Brown / Rebecca Sitsapesan / Phillip J Stansfeld / Juha T Huiskonen / Paolo Tammaro / Alessio Accardi / Elisabeth P Carpenter /
Abstract: Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic ...Membranes in cells have defined distributions of lipids in each leaflet, controlled by lipid scramblases and flip/floppases. However, for some intracellular membranes such as the endoplasmic reticulum (ER) the scramblases have not been identified. Members of the TMEM16 family have either lipid scramblase or chloride channel activity. Although TMEM16K is widely distributed and associated with the neurological disorder autosomal recessive spinocerebellar ataxia type 10 (SCAR10), its location in cells, function and structure are largely uncharacterised. Here we show that TMEM16K is an ER-resident lipid scramblase with a requirement for short chain lipids and calcium for robust activity. Crystal structures of TMEM16K show a scramblase fold, with an open lipid transporting groove. Additional cryo-EM structures reveal extensive conformational changes from the cytoplasmic to the ER side of the membrane, giving a state with a closed lipid permeation pathway. Molecular dynamics simulations showed that the open-groove conformation is necessary for scramblase activity.
History
DepositionMar 26, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 1, 2019Provider: repository / Type: Initial release
Revision 1.1Jul 10, 2019Group: Data collection / Category: diffrn_source / Item: _diffrn_source.pdbx_synchrotron_site
Revision 1.2Oct 16, 2019Group: Data collection / Category: reflns / reflns_shell / Item: _reflns.pdbx_diffrn_id / _reflns_shell.pdbx_diffrn_id
Revision 1.3Nov 20, 2019Group: 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 1.4Jan 24, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_initial_refinement_model / struct_conn
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _struct_conn.pdbx_dist_value / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Anoctamin-10
B: Anoctamin-10
hetero molecules


Theoretical massNumber of molelcules
Total (without water)154,7938
Polymers154,5522
Non-polymers2406
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3520 Å2
ΔGint-91 kcal/mol
Surface area57310 Å2
MethodPISA
2


  • Idetical with deposited unit
  • defined by author&software
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area3520 Å2
ΔGint-91 kcal/mol
Surface area57310 Å2
MethodPISA
Unit cell
Length a, b, c (Å)113.158, 152.267, 153.703
Angle α, β, γ (deg.)90.000, 90.000, 90.000
Int Tables number19
Space group name H-MP212121

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Components

#1: Protein Anoctamin-10 / Calcium-dependent chloride channel / Transmembrane protein 16K


Mass: 77276.016 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: ANO10, TMEM16K / Plasmid: PFB-CT10HF-LIC / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9NW15
#2: Chemical
ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Ca

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Experimental details

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Experiment

ExperimentMethod: X-RAY DIFFRACTION / Number of used crystals: 1

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Sample preparation

CrystalDensity Matthews: 4.34 Å3/Da / Density % sol: 71.66 %
Crystal growTemperature: 293 K / Method: vapor diffusion, sitting drop / pH: 7
Details: 0.1M calcium acetate 0.1M HEPES pH 7.0 22% PEG400 1CMC C12E9

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Data collection

DiffractionMean temperature: 100 K / Serial crystal experiment: N
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.9686 Å
DetectorType: DECTRIS PILATUS3 S 6M / Detector: PIXEL / Date: Nov 29, 2015
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9686 Å / Relative weight: 1
Reflection

Entry-ID: 6R65

Resolution (Å)Num. obs% possible obs (%)Redundancy (%)CC1/2Rmerge(I) obsRpim(I) allRrim(I) allDiffraction-IDNet I/σ(I)
3.497-78.1962623776.43.60.9980.130.120.17817.1
3.5-78.193401099.63.710.1720.1030.20125.6
Reflection shell
Resolution (Å)Redundancy (%)Rmerge(I) obsMean I/σ(I) obsNum. unique obsCC1/2Rpim(I) allRrim(I) allDiffraction-ID% possible all
3.5-3.6063.50.7031.713120.5080.6510.96144.1
3.5-3.593.81.8940.824940.3521.1042.2299.8

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Processing

Software
NameVersionClassification
BUSTER2.10.3refinement
PDB_EXTRACT3.24data extraction
XDSVERSION Oct 15, 2015data reduction
XDSVERSION Oct 15, 2015data scaling
STARANISO1.0.5data scaling
PHASER2.6.1phasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 5OC9

5oc9


Resolution: 3.5→39.69 Å / Cor.coef. Fo:Fc: 0.817 / Cor.coef. Fo:Fc free: 0.794 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.605
Details: Structure refined using STARANISO anistropically truncated data. Reference model (LSSR) restraints to PDB:5OC9 were also used
RfactorNum. reflection% reflectionSelection details
Rfree0.249 1191 4.99 %RANDOM
Rwork0.237 ---
obs0.238 23883 70 %-
Displacement parametersBiso max: 229.57 Å2 / Biso mean: 92.15 Å2 / Biso min: 57.28 Å2
Baniso -1Baniso -2Baniso -3
1-13.8649 Å20 Å20 Å2
2---14.5408 Å20 Å2
3---0.6758 Å2
Refine analyzeLuzzati coordinate error obs: 0.59 Å
Refinement stepCycle: final / Resolution: 3.5→39.69 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9642 0 6 0 9648
Biso mean--80.44 --
Num. residues----1234
Refine LS restraints
Refine-IDTypeNumberRestraint functionWeightDev ideal
X-RAY DIFFRACTIONt_dihedral_angle_d4418SINUSOIDAL2
X-RAY DIFFRACTIONt_trig_c_planes
X-RAY DIFFRACTIONt_gen_planes1636HARMONIC5
X-RAY DIFFRACTIONt_it9878HARMONIC20
X-RAY DIFFRACTIONt_nbd0SEMIHARMONIC5
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_chiral_improper_torsion1312SEMIHARMONIC5
X-RAY DIFFRACTIONt_sum_occupancies
X-RAY DIFFRACTIONt_utility_distance
X-RAY DIFFRACTIONt_utility_angle
X-RAY DIFFRACTIONt_utility_torsion
X-RAY DIFFRACTIONt_ideal_dist_contact11593SEMIHARMONIC4
X-RAY DIFFRACTIONt_bond_d9878HARMONIC20.008
X-RAY DIFFRACTIONt_angle_deg13455HARMONIC20.9
X-RAY DIFFRACTIONt_omega_torsion1.95
X-RAY DIFFRACTIONt_other_torsion2.8
LS refinement shellResolution: 3.5→3.65 Å / Rfactor Rfree error: 0 / Total num. of bins used: 12
RfactorNum. reflection% reflection
Rfree0.2363 68 4.74 %
Rwork0.2281 1367 -
all0.2285 1435 -
obs--34.79 %
Refinement TLS params.

Method: refined / Refine-ID: X-RAY DIFFRACTION

IDL11 (°2)L12 (°2)L13 (°2)L22 (°2)L23 (°2)L33 (°2)S11 (Å °)S12 (Å °)S13 (Å °)S21 (Å °)S22 (Å °)S23 (Å °)S31 (Å °)S32 (Å °)S33 (Å °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
12.6263-0.57960.4781.5143-0.72022.02180.105-0.0445-0.2020.1074-0.05620.03350.1624-0.0905-0.0487-0.0680.03280.0447-0.27950.0285-0.2285-55.19472.1671-20.6089
21.6155-0.0469-0.09470.7248-0.63642.21190.10040.15390.1680.05990.0536-0.0121-0.04320.0361-0.154-0.06920.0787-0.0092-0.2765-0.0103-0.2074-9.14126.4423-14.8697
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection detailsAuth asym-IDAuth seq-ID
1X-RAY DIFFRACTION1{A|14 - 703}A14 - 703
2X-RAY DIFFRACTION2{B|15 - 703}B15 - 703

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