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- PDB-7sqh: Structure of the human proton-activated chloride channel ASOR in ... -

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Basic information

Entry
Database: PDB / ID: 7sqh
TitleStructure of the human proton-activated chloride channel ASOR in desensitized conformation
ComponentsProton-activated chloride channel
KeywordsMEMBRANE PROTEIN / ion channel / chloride channel
Function / homologypH-gated chloride channel activity / TMEM206 protein / TMEM206 protein family / chloride transport / chloride channel complex / cell surface / plasma membrane / Proton-activated chloride channel
Function and homology information
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.5 Å
AuthorsLong, S.B. / Wang, C. / Delgado, B.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131921 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30CA008748 United States
CitationJournal: Sci Adv / Year: 2022
Title: Gating choreography and mechanism of the human proton-activated chloride channel ASOR.
Authors: Chongyuan Wang / Maya M Polovitskaya / Bryce D Delgado / Thomas J Jentsch / Stephen B Long /
Abstract: The proton-activated chloride channel ASOR (TMEM206/PAC) permeates anions across cellular membranes in response to acidification, thereby enhancing acid-induced cell death and regulating endocytosis. ...The proton-activated chloride channel ASOR (TMEM206/PAC) permeates anions across cellular membranes in response to acidification, thereby enhancing acid-induced cell death and regulating endocytosis. The molecular mechanisms of pH-dependent control are not understood, in part because structural information for an activated conformation of ASOR is lacking. Here, we reconstitute function from purified protein and present a 3.1-Å-resolution cryo-electron microscopy structure of human ASOR at acidic pH in an activated conformation. The work contextualizes a previous acidic pH structure as a desensitized conformation. Combined with electrophysiological studies and high-resolution structures of resting and desensitized states, the work reveals mechanisms of proton sensing and ion pore gating. Clusters of extracellular acidic residues function as pH sensors and coalesce when protonated. Ensuing conformational changes induce metamorphosis of transmembrane helices to fashion an ion conduction pathway unique to the activated conformation. The studies identify a new paradigm of channel gating in this ubiquitous ion channel.
History
DepositionNov 5, 2021Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 23, 2022Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: Proton-activated chloride channel
B: Proton-activated chloride channel
C: Proton-activated chloride channel
hetero molecules


Theoretical massNumber of molelcules
Total (without water)122,93115
Polymers120,2763
Non-polymers2,65412
Water37821
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein Proton-activated chloride channel / hPAC / Acid-sensitive outwardly-rectifying anion channel / ASOR / Proton-activated outwardly ...hPAC / Acid-sensitive outwardly-rectifying anion channel / ASOR / Proton-activated outwardly rectifying anion channel / PAORAC / Transmembrane protein 206 / hTMEM206


Mass: 40092.047 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: PACC1, C1orf75, TMEM206 / Production host: Homo sapiens (human) / References: UniProt: Q9H813
#2: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 21 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN

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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: proton activated chloride channel TMEM206 in resting conformation
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: Expi293, Invitrogen
Buffer solutionpH: 4.5
Buffer component
IDConc.NameFormulaBuffer-ID
15 mMHEPES1
2150 mMsodium chlorideNaCl1
350 mMcitrate1
SpecimenConc.: 1.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Monodisperse sample
Specimen supportGrid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 298 K / Details: 2 second blot, blot force of 0

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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: BRIGHT FIELD / Nominal magnification: 22500 X / Nominal defocus max: -3000 nm / Nominal defocus min: -1000 nm / Alignment procedure: BASIC
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 71 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 157906
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.19.1_4122: / Classification: refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4Gctf1.06CTF correction
7Coot0.9.5model fitting
9PHENIX1.19.1-4122model refinement
10cryoSPARCv3.1.0initial Euler assignment
11RELION3.1initial Euler assignment
12cryoSPARCv3.1.0final Euler assignment
13cryoSPARCv3.1.0classification
14cryoSPARCv3.1.03D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3097510
3D reconstructionResolution: 2.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 157906 / Symmetry type: POINT
Atomic model buildingB value: 30 / Protocol: AB INITIO MODEL / Space: REAL / Target criteria: Correlation coefficient
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0037092
ELECTRON MICROSCOPYf_angle_d0.559600
ELECTRON MICROSCOPYf_dihedral_angle_d9.565984
ELECTRON MICROSCOPYf_chiral_restr0.0441098
ELECTRON MICROSCOPYf_plane_restr0.0031200

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