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- PDB-8ta4: Cryo-EM structure of the human CLC-2 chloride channel transmembra... -

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Basic information

Entry
Database: PDB / ID: 8ta4
TitleCryo-EM structure of the human CLC-2 chloride channel transmembrane domain with symmetric C-terminal
ComponentsChloride channel protein 2
KeywordsTRANSPORT PROTEIN / Chloride / Channel / Inhibitor / Protein / Voltage gated
Function / homology
Function and homology information


regulation of aldosterone biosynthetic process / cell differentiation involved in salivary gland development / astrocyte end-foot / acinar cell differentiation / voltage-gated chloride channel activity / dendritic spine membrane / chloride transport / phagocytosis, engulfment / positive regulation of oligodendrocyte differentiation / chloride channel complex ...regulation of aldosterone biosynthetic process / cell differentiation involved in salivary gland development / astrocyte end-foot / acinar cell differentiation / voltage-gated chloride channel activity / dendritic spine membrane / chloride transport / phagocytosis, engulfment / positive regulation of oligodendrocyte differentiation / chloride channel complex / lung development / Stimuli-sensing channels / myelin sheath / retina development in camera-type eye / basolateral plasma membrane / perikaryon / postsynaptic membrane / axon / plasma membrane
Similarity search - Function
Chloride channel ClC-2 / : / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / CBS domain superfamily / CBS domain profile.
Similarity search - Domain/homology
Chloride channel protein 2
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.75 Å
AuthorsXu, M. / Neelands, T. / Powers, A.S. / Liu, Y. / Miller, S. / Pintilie, G. / Du Bois, J. / Dror, R.O. / Chiu, W. / Maduke, M.
Funding support United States, 4items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM079429 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM129541 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS113611 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)NS125767 United States
CitationJournal: Elife / Year: 2024
Title: CryoEM structures of the human CLC-2 voltage-gated chloride channel reveal a ball-and-chain gating mechanism.
Authors: Mengyuan Xu / Torben Neelands / Alexander S Powers / Yan Liu / Steven D Miller / Grigore D Pintilie / J Du Bois / Ron O Dror / Wah Chiu / Merritt Maduke /
Abstract: CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated ...CLC-2 is a voltage-gated chloride channel that contributes to electrical excitability and ion homeostasis in many different tissues. Among the nine mammalian CLC homologs, CLC-2 is uniquely activated by hyperpolarization, rather than depolarization, of the plasma membrane. The molecular basis for the divergence in polarity of voltage gating among closely related homologs has been a long-standing mystery, in part because few CLC channel structures are available. Here, we report cryoEM structures of human CLC-2 at 2.46 - 2.76 Å, in the presence and absence of the selective inhibitor AK-42. AK-42 binds within the extracellular entryway of the Cl-permeation pathway, occupying a pocket previously proposed through computational docking studies. In the apo structure, we observed two distinct conformations involving rotation of one of the cytoplasmic C-terminal domains (CTDs). In the absence of CTD rotation, an intracellular N-terminal 15-residue hairpin peptide nestles against the TM domain to physically occlude the Cl-permeation pathway. This peptide is highly conserved among species variants of CLC-2 but is not present in other CLC homologs. Previous studies suggested that the N-terminal domain of CLC-2 influences channel properties via a "ball-and-chain" gating mechanism, but conflicting data cast doubt on such a mechanism, and thus the structure of the N-terminal domain and its interaction with the channel has been uncertain. Through electrophysiological studies of an N-terminal deletion mutant lacking the 15-residue hairpin peptide, we support a model in which the N-terminal hairpin of CLC-2 stabilizes a closed state of the channel by blocking the cytoplasmic Cl-permeation pathway.
History
DepositionJun 26, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 31, 2024Provider: repository / Type: Initial release
Revision 1.1Feb 28, 2024Group: 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.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 2.0Mar 20, 2024Group: Advisory / Atomic model ...Advisory / Atomic model / Author supporting evidence / Data collection / Derived calculations / Refinement description / Structure summary
Category: atom_site / em_software ...atom_site / em_software / pdbx_contact_author / pdbx_entity_instance_feature / pdbx_entry_details / pdbx_struct_assembly / pdbx_struct_assembly_prop / pdbx_struct_sheet_hbond / pdbx_validate_close_contact / pdbx_validate_planes / pdbx_validate_rmsd_angle / pdbx_validate_rmsd_bond / pdbx_validate_torsion / refine_ls_restr / struct_conf / struct_sheet / struct_sheet_order / struct_sheet_range
Item: _pdbx_entry_details.has_ligand_of_interest / _pdbx_struct_assembly.details ..._pdbx_entry_details.has_ligand_of_interest / _pdbx_struct_assembly.details / _pdbx_struct_assembly.method_details / _struct_sheet.number_strands
Description: Model completeness / Provider: author / Type: Coordinate replacement

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Chloride channel protein 2
B: Chloride channel protein 2
hetero molecules


Theoretical massNumber of molelcules
Total (without water)197,3564
Polymers197,2852
Non-polymers712
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8630 Å2
ΔGint-95 kcal/mol
Surface area50750 Å2
MethodPISA

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Components

#1: Protein Chloride channel protein 2 / ClC-2


Mass: 98642.352 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLCN2 / Production host: Homo sapiens (human) / References: UniProt: P51788
#2: Chemical ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Chloride channel protein 2 / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingAverage exposure time: 5.6 sec. / Electron dose: 50 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of real images: 14300

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Processing

EM software
IDNameVersionCategory
1cryoSPARC3.2particle selection
2EPU2image acquisition
4cryoSPARC3.2CTF correction
7Coot0.9model fitting
9UCSF ChimeraX1.6model refinement
10cryoSPARC3.2initial Euler assignment
11cryoSPARC3.2final Euler assignment
12cryoSPARC3.2classification
13cryoSPARC3.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 5214695
3D reconstructionResolution: 2.75 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56580 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Q-score
Atomic model buildingAccession code: 6qvc / Source name: SwissModel / Type: in silico model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0059959
ELECTRON MICROSCOPYf_angle_d0.60113527
ELECTRON MICROSCOPYf_dihedral_angle_d3.9121339
ELECTRON MICROSCOPYf_chiral_restr0.0411586
ELECTRON MICROSCOPYf_plane_restr0.0051675

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