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- PDB-6r7x: CryoEM structure of calcium-bound human TMEM16K / Anoctamin 10 in... -

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Entry
Database: PDB / ID: 6r7x
TitleCryoEM structure of calcium-bound human TMEM16K / Anoctamin 10 in detergent (2mM Ca2+, closed form)
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


Stimuli-sensing channels / intracellular calcium activated chloride channel activity / calcium activated cation channel activity / chloride transport / cation transport / ion transmembrane transport / membrane / integral component of membrane / plasma membrane
Anoctamin / Anoctamin-10 / Calcium-activated chloride channel
Anoctamin-10
Specimen sourceHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.47 Å
AuthorsPike, A.C.W. / Bushell, S.R. / Shintre, C.A. / Tessitore, A. / Baronina, A. / Chu, A. / Mukhopadhyay, S. / Shrestha, L. / Chalk, R. / Burgess-Brown, N.A. / Love, J. / Huiskonen, J.T. / Edwards, A.M. / Arrowsmith, C.H. / Bountra, C. / Carpenter, E.P. / Structural Genomics Consortium (SGC)
Funding supportUnited Kingdom , 2件
OrganizationGrant numberCountry
Wellcome Trust106169/Z/14/ZUnited Kingdom
European Commission115766United Kingdom
CitationJournal: TO BE PUBLISHED
Title: CryoEM structure of calcium-bound human TMEM16K / Anoctamin 10 in detergent (closed form)
Authors: Pike, A.C.W. / Bushell, S.R. / Shintre, C.A. / Tessitore, A. / Baronina, A. / Chu, A. / Mukhopadhyay, S. / Shrestha, L. / Chalk, R. / Burgess-Brown, N.A. / Love, J. / Huiskonen, J.T. / Edwards, A.M. / Arrowsmith, C.H. / Bountra, C. / Carpenter, E.P. / Structural Genomics Consortium (SGC)
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Mar 29, 2019 / Release: May 1, 2019Array

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Structure visualization

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Assembly

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


Theoretical massNumber of molelcules
Total (without water)166,30530
Polymers154,5522
Non-polymers11,75328
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area17910 Å2
ΔGint-345 kcal/mol
Surface area58640 Å2
MethodPISA

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Components

#1: Protein/peptide 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 / Calcium
#3: Chemical ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE


Mass: 790.145 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid *YM
#4: Chemical
ChemComp-UMQ / UNDECYL-MALTOSIDE / UNDECYL-BETA-D-MALTOPYRANOSIDE


Mass: 496.589 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: C23H44O11 / Comment: detergent *YM

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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: Anoctamin 10Calcium-dependent chloride channel / Type: ORGANELLE OR CELLULAR COMPONENT / Entity ID: 1 / Source: RECOMBINANT
Molecular weightValue: 0.153 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm) / Plasmid: PFB-CT10HF-LIC
Buffer solutionpH: 7.5
Buffer component

Buffer-ID: 1

IDConc.NameFormula
1150 mMsodium chlorideNaCl
220 mMNa HEPESC8H18N2O4S
30.045 % (w/v)Undecyl b-D MaltopyranosideC23H44O11
40.0045 % (w/v)cholesteryl hemisuccinateC31H50O4
52 mMcalcium chlorideCaCl2
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K / Details: blot for 3.5sec prior to plunging

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1250 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µns / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 8 sec. / Electron dose: 50.9 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 3421
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansMovie frames/image: 20 / Used frames/image: 1-20

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
EM software
IDNameVersionCategory
1Gautomatch0.53particle selection
2EPUimage acquisition
4CTFFIND4.1.5CTF correction
7Cootmodel fitting
9RELION3initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 300992
Details: Particles count after a single round of 2D classification
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.47 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 70940 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingB value: 176 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coef
Details: phenix.real_space_refine with NCS contraints, rotamer, cbeta and ramachandran restraints using postprocessed RELION3 map
Atomic model buildingPDB-ID: 5OC9
Pdb chain-ID: A

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