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- PDB-9o95: Cryo-EM structure of CLC-ec1 at pH 7.5 -

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Basic information

Entry
Database: PDB / ID: 9o95
TitleCryo-EM structure of CLC-ec1 at pH 7.5
ComponentsH(+)/Cl(-) exchange transporter ClcA
KeywordsTRANSPORT PROTEIN / transmembrane protein cryo-EM Antiporter
Function / homology
Function and homology information


cellular stress response to acidic pH / chloride:proton antiporter activity / voltage-gated chloride channel activity / proton transmembrane transport / chloride transmembrane transport / identical protein binding / plasma membrane
Similarity search - Function
Chloride channel, ClcA / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel
Similarity search - Domain/homology
H(+)/Cl(-) exchange transporter ClcA
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsChien, C.-T. / Chiu, W. / Maduke, M.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM113195 United States
CitationJournal: Nat Commun / Year: 2026
Title: Molecular mechanism of exchange coupling in CLC chloride/proton antiporters.
Authors: Deniz Aydin / Chih-Ta Chien / Jürgen Kreiter / Amy R Nava / Jasmina M Portasikova / Lukas Fojtik / Briana L Sobecks / Catalina Mosquera / Petr Man / Ron O Dror / Wah Chiu / Merritt Maduke /
Abstract: The ubiquitous CLC membrane transporters are unique in their ability to exchange anions for cations. Despite extensive study, there is no mechanistic model that fully explains their 2:1 Cl/H ...The ubiquitous CLC membrane transporters are unique in their ability to exchange anions for cations. Despite extensive study, there is no mechanistic model that fully explains their 2:1 Cl/H stoichiometric exchange mechanism. Here, we provide such a model. Using differential hydrogen-deuterium exchange mass spectrometry, cryo-EM structure determination, and molecular dynamics simulations, we uncovered conformational dynamics in CLC-ec1, a bacterial CLC homolog that has served as a paradigm for this family of transporters. Simulations based on a cryo-EM structure at pH 3 revealed critical steps in the transport mechanism, including release of Cl ions to the extracellular side, opening of the inner gate, and water wires that facilitate H transport. Surprisingly, these water wires occurred independently of Cl binding, prompting us to reassess the relationship between Cl binding and Cl/H coupling. Using isothermal titration calorimetry and quantitative flux assays on mutants with reduced Cl binding affinity, we conclude that, while Cl binding is necessary for coupling, even weak binding can support Cl/H coupling. By integrating our findings with existing literature, we establish a complete and efficient CLC 2:1 Cl/H exchange mechanism.
History
DepositionApr 17, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 7, 2026Provider: 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)104,3736
Polymers104,2312
Non-polymers1424
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein H(+)/Cl(-) exchange transporter ClcA / ClC-ec1


Mass: 52115.359 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: clcA, eriC, yadQ, b0155, JW5012 / Production host: Escherichia coli (E. coli) / References: UniProt: P37019
#2: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl
Has ligand of interestN
Has protein modificationN

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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: H(+)/Cl(-) exchange transporter ClcA / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia coli (E. coli)
Source (recombinant)Organism: Escherichia coli (E. coli)
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: TFS 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: 800 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21.1_5286: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 72103 / Symmetry type: POINT

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