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- PDB-5oc9: Crystal Structure of human TMEM16K / Anoctamin 10 -

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Basic information

Entry
Database: PDB / ID: 5oc9
TitleCrystal Structure of human TMEM16K / Anoctamin 10
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
(2R)-2,3-dihydroxypropyl (7Z)-hexadec-7-enoate / Anoctamin-10
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodX-RAY DIFFRACTION / SYNCHROTRON / MOLECULAR REPLACEMENT / Resolution: 3.2 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, 1items
OrganizationGrant numberCountry
Wellcome Trust 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
DepositionJun 29, 2017Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 25, 2018Provider: repository / Type: Initial release
Revision 1.1Jun 12, 2019Group: Data collection / Structure summary
Category: audit_author / database_PDB_rev / database_PDB_rev_record
Item: _audit_author.name
Revision 1.2Nov 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 1.3Jan 17, 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)160,74527
Polymers154,5522
Non-polymers6,19325
Water1,00956
1


  • Idetical with deposited unit
  • defined by software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area4950 Å2
ΔGint-107 kcal/mol
Surface area56670 Å2
MethodPISA
Unit cell
Length a, b, c (Å)59.150, 153.590, 218.780
Angle α, β, γ (deg.)90.00, 90.00, 90.00
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.: 7 / Source method: obtained synthetically / Formula: Ca
#3: Chemical
ChemComp-79M / (2R)-2,3-dihydroxypropyl (7Z)-hexadec-7-enoate / [(2R)-2,3-bis(oxidanyl)propyl] (Z)-hexadec-7-enoate


Mass: 328.487 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: C19H36O4
#4: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 56 / Source method: isolated from a natural source / Formula: H2O

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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: 3.22 Å3/Da / Density % sol: 61.81 %
Crystal growTemperature: 293 K / Method: lipidic cubic phase / pH: 6
Details: 0.1M MES pH 6.0 -- 0.1M sodium chloride -- 0.1M calcium chloride -- 30% PEG300

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Data collection

DiffractionMean temperature: 100 K
Diffraction sourceSource: SYNCHROTRON / Site: Diamond / Beamline: I24 / Wavelength: 0.9686 Å
DetectorType: DECTRIS PILATUS3 6M / Detector: PIXEL / Date: Jul 17, 2016
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthWavelength: 0.9686 Å / Relative weight: 1
ReflectionResolution: 3.2→76.79 Å / Num. obs: 33892 / % possible obs: 99.9 % / Redundancy: 5.1 % / Biso Wilson estimate: 43.191 Å2 / CC1/2: 0.99 / Rmerge(I) obs: 0.285 / Rpim(I) all: 0.139 / Net I/σ(I): 5.9
Reflection shellResolution: 3.2→3.28 Å / Redundancy: 5.2 % / Rmerge(I) obs: 1.265 / Mean I/σ(I) obs: 1.4 / Num. unique obs: 2488 / CC1/2: 0.535 / Rpim(I) all: 0.61 / % possible all: 99.9

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Processing

Software
NameVersionClassification
BUSTER2.10.2refinement
XDSdata reduction
Aimlessdata scaling
MrBUMPphasing
MR-Rosettaphasing
RefinementMethod to determine structure: MOLECULAR REPLACEMENT
Starting model: 4WIS

4wis


Resolution: 3.2→76.79 Å / Cor.coef. Fo:Fc: 0.8549 / Cor.coef. Fo:Fc free: 0.855 / Cross valid method: THROUGHOUT / σ(F): 0 / SU Rfree Blow DPI: 0.42
Details: Refined with BUSTER with single TLS group per chain
RfactorNum. reflection% reflectionSelection details
Rfree0.2452 1699 5.02 %RANDOM
Rwork0.2283 ---
obs0.2292 33824 99.91 %-
Displacement parametersBiso mean: 57.98 Å2
Baniso -1Baniso -2Baniso -3
1--16.5833 Å20 Å20 Å2
2--5.8188 Å20 Å2
3---10.7645 Å2
Refine analyzeLuzzati coordinate error obs: 0.485 Å
Refinement stepCycle: 1 / Resolution: 3.2→76.79 Å
ProteinNucleic acidLigandSolventTotal
Num. atoms9806 0 276 56 10138
Refine LS restraints
Refine-IDTypeDev idealNumberRestraint functionWeight
X-RAY DIFFRACTIONt_bond_d0.00810295HARMONIC2
X-RAY DIFFRACTIONt_angle_deg0.8713881HARMONIC2
X-RAY DIFFRACTIONt_dihedral_angle_d4785SINUSOIDAL2
X-RAY DIFFRACTIONt_incorr_chiral_ct
X-RAY DIFFRACTIONt_pseud_angle
X-RAY DIFFRACTIONt_trig_c_planes199HARMONIC2
X-RAY DIFFRACTIONt_gen_planes1469HARMONIC5
X-RAY DIFFRACTIONt_it10295HARMONIC20
X-RAY DIFFRACTIONt_nbd1SEMIHARMONIC5
X-RAY DIFFRACTIONt_omega_torsion1.94
X-RAY DIFFRACTIONt_other_torsion3.05
X-RAY DIFFRACTIONt_improper_torsion
X-RAY DIFFRACTIONt_chiral_improper_torsion1311SEMIHARMONIC5
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_contact12150SEMIHARMONIC4
LS refinement shellResolution: 3.2→3.3 Å / Total num. of bins used: 17
RfactorNum. reflection% reflection
Rfree0.3086 138 4.76 %
Rwork0.247 2764 -
all0.25 2902 -
obs--99.9 %
Refinement TLS params.

S33: -0.0003 Å ° / 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 °)T11 (Å2)T12 (Å2)T13 (Å2)T22 (Å2)T23 (Å2)T33 (Å2)Origin x (Å)Origin y (Å)Origin z (Å)
10.7338-0.1906-0.20131.45310.47250.9163-0.00510.15940.023-0.00940.0054-0.04630.0274-0.0604-0.0657-0.00320.0064-0.1601-0.0406-0.185739.840884.865854.5677
20.5677-0.0054-0.01010.6327-0.23491.24150.013-0.08290.0106-0.026-0.01280.01890.13430.0281-0.0109-0.0480.0172-0.1842-0.0232-0.15654.706691.84597.2179
Refinement TLS group
IDRefine-IDRefine TLS-IDSelection details
1X-RAY DIFFRACTION1{A|14 - 802}
2X-RAY DIFFRACTION2{B|14 - 803}

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