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- PDB-7n0k: Cryo-EM structure of TACAN in the apo form (TMEM120A) -

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Basic information

Entry
Database: PDB / ID: 7n0k
TitleCryo-EM structure of TACAN in the apo form (TMEM120A)
ComponentsIon channel TACAN
KeywordsMEMBRANE PROTEIN / lipid metabolism / coenzyme A
Function / homology
Function and homology information


protein heterooligomerization / nuclear inner membrane / fat cell differentiation / detection of mechanical stimulus involved in sensory perception of pain / ion transmembrane transport / protein homooligomerization / ion channel activity / membrane / integral component of membrane / plasma membrane
Similarity search - Function
Ion channel TACAN/TMEM120B / TMPIT-like protein
Similarity search - Domain/homology
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsNiu, Y. / Tao, X. / MacKinnon, R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM43949 United States
CitationJournal: Elife / Year: 2021
Title: Analysis of the mechanosensor channel functionality of TACAN.
Authors: Yiming Niu / Xiao Tao / George Vaisey / Paul Dominic B Olinares / Hanan Alwaseem / Brian T Chait / Roderick MacKinnon /
Abstract: Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the ...Mechanosensitive ion channels mediate transmembrane ion currents activated by mechanical forces. A mechanosensitive ion channel called TACAN was recently reported. We began to study TACAN with the intent to understand how it senses mechanical forces and functions as an ion channel. Using cellular patch-recording methods, we failed to identify mechanosensitive ion channel activity. Using membrane reconstitution methods, we found that TACAN, at high protein concentrations, produces heterogeneous conduction levels that are not mechanosensitive and are most consistent with disruptions of the lipid bilayer. We determined the structure of TACAN using single-particle cryo-electron microscopy and observed that it is a symmetrical dimeric transmembrane protein. Each protomer contains an intracellular-facing cleft with a coenzyme A cofactor, confirmed by mass spectrometry. The TACAN protomer is related in three-dimensional structure to a fatty acid elongase, ELOVL7. Whilst its physiological function remains unclear, we anticipate that TACAN is not a mechanosensitive ion channel.
History
DepositionMay 25, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 15, 2021Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
B: Ion channel TACAN
A: Ion channel TACAN


Theoretical massNumber of molelcules
Total (without water)81,5952
Polymers81,5952
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: mass spectrometry
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area6360 Å2
ΔGint-60 kcal/mol
Surface area34130 Å2

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Components

#1: Protein Ion channel TACAN / Transmembrane protein 120A


Mass: 40797.297 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Tmem120a, Net29, Tacan / Production host: Homo sapiens (human) / References: UniProt: Q8C1E7

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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: Homo-dimeric assembly of wild type TACAN(TMEM120A) / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: KILODALTONS/NANOMETER / Experimental value: NO
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.4
Details: 20 mM HEPES pH 7.4, 250 mM NaCl, and 0.06% Digitonin (w/v)
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 298 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: FLOOD BEAM
Electron lensMode: DARK FIELD / C2 aperture diameter: 100 µm
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 75.4 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1

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Processing

SoftwareName: PHENIX / Version: dev_3751: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctf1.0.6CTF correction
7UCSF Chimera1.14model fitting
8Coot0.9model fitting
9PHENIX1.151.2model fitting
11RELION3initial Euler assignment
12RELION3.1initial Euler assignment
13cryoSPARC2.9.0initial Euler assignment
14RELION3.1final Euler assignment
15RELION3.1classification
16cryoSPARC2.9.03D reconstruction
17PHENIX1.151.2model refinement
CTF correctionType: NONE
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110090 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0035304
ELECTRON MICROSCOPYf_angle_d0.4827176
ELECTRON MICROSCOPYf_dihedral_angle_d14.395680
ELECTRON MICROSCOPYf_chiral_restr0.031774
ELECTRON MICROSCOPYf_plane_restr0.003890

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