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- PDB-8gah: CLC-ec1 L25C/A450C/C85A at pH 4.5 100mM Cl Twist -

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Basic information

Entry
Database: PDB / ID: 8gah
TitleCLC-ec1 L25C/A450C/C85A at pH 4.5 100mM Cl Twist
ComponentsH(+)/Cl(-) exchange transporter ClcA
KeywordsTRANSPORT PROTEIN / CLC-ec1 / ecCLC / eriC / CLC transporter / chloride proton antiporter
Function / homologyChloride channel, ClcA / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / voltage-gated chloride channel activity / antiporter activity / plasma membrane / H(+)/Cl(-) exchange transporter ClcA
Function and homology information
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsFortea, E. / Lee, S. / Argyos, Y. / Chadda, R. / Ciftci, D. / Huysmans, G. / Robertson, J.L. / Boudker, O. / Accardi, A.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01 GM128420 United States
CitationJournal: Nat Struct Mol Biol / Year: 2024
Title: Structural basis of pH-dependent activation in a CLC transporter.
Authors: Eva Fortea / Sangyun Lee / Rahul Chadda / Yiorgos Argyros / Priyanka Sandal / Robyn Mahoney-Kruszka / Hatice Didar Ciftci / Maria E Falzone / Gerard Huysmans / Janice L Robertson / Olga ...Authors: Eva Fortea / Sangyun Lee / Rahul Chadda / Yiorgos Argyros / Priyanka Sandal / Robyn Mahoney-Kruszka / Hatice Didar Ciftci / Maria E Falzone / Gerard Huysmans / Janice L Robertson / Olga Boudker / Alessio Accardi /
Abstract: CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure ...CLCs are dimeric chloride channels and anion/proton exchangers that regulate processes such as muscle contraction and endo-lysosome acidification. Common gating controls their activity; its closure simultaneously silences both protomers, and its opening allows them to independently transport ions. Mutations affecting common gating in human CLCs cause dominant genetic disorders. The structural rearrangements underlying common gating are unknown. Here, using single-particle cryo-electron microscopy, we show that the prototypical Escherichia coli CLC-ec1 undergoes large-scale rearrangements in activating conditions. The slow, pH-dependent remodeling of the dimer interface leads to the concerted opening of the intracellular H pathways and is required for transport. The more frequent formation of short water wires in the open H pathway enables Cl pore openings. Mutations at disease-causing sites favor CLC-ec1 activation and accelerate common gate opening in the human CLC-7 exchanger. We suggest that the pH activation mechanism of CLC-ec1 is related to the common gating of CLC-7.
History
DepositionFeb 22, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 7, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: H(+)/Cl(-) exchange transporter ClcA
B: H(+)/Cl(-) exchange transporter ClcA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)98,5506
Polymers98,4082
Non-polymers1424
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein H(+)/Cl(-) exchange transporter ClcA


Mass: 49204.141 Da / Num. of mol.: 2 / Mutation: L25C,C85A,A450C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: yadQ / Production host: Escherichia coli (E. coli) / References: UniProt: J7Q633
#2: Chemical
ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 4 / 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: Structure of ecCLC-L25C/A450C/C85A mutant at pH 4.5 in 100mM Cl
Type: CELL / Entity ID: #1 / Source: NATURAL
Source (natural)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 4.5
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 FIELDBright-field microscopy / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm
Image recordingElectron dose: 58.653 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.14_3260refinement
PHENIX1.14_3260refinement
EM softwareName: cryoSPARC / Version: 3.3.2 / Category: particle selection
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 429018 / Symmetry type: POINT
RefinementStereochemistry target values: CDL v1.2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00526563
ELECTRON MICROSCOPYf_angle_d0.86928920
ELECTRON MICROSCOPYf_chiral_restr0.04871056
ELECTRON MICROSCOPYf_plane_restr0.00781116
ELECTRON MICROSCOPYf_dihedral_angle_d12.44143783

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