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- PDB-9do0: Human ClC-3 -

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Basic information

Entry
Database: PDB / ID: 9do0
TitleHuman ClC-3
ComponentsH(+)/Cl(-) exchange transporter 3
KeywordsMEMBRANE PROTEIN / Ion channel / lysosomal protein / CLC
Function / homology
Function and homology information


volume-sensitive chloride channel activity / chloride:proton antiporter activity / synaptic vesicle lumen acidification / negative regulation of cell volume / endosomal lumen acidification / regulation of pH / voltage-gated chloride channel activity / specific granule / photoreceptor cell maintenance / vesicle membrane ...volume-sensitive chloride channel activity / chloride:proton antiporter activity / synaptic vesicle lumen acidification / negative regulation of cell volume / endosomal lumen acidification / regulation of pH / voltage-gated chloride channel activity / specific granule / photoreceptor cell maintenance / vesicle membrane / antiporter activity / phagocytosis, engulfment / synaptic transmission, GABAergic / chloride channel activity / positive regulation of reactive oxygen species biosynthetic process / phagocytic vesicle / axon terminus / chloride transmembrane transport / secretory granule / adult locomotory behavior / synaptic transmission, glutamatergic / PDZ domain binding / recycling endosome / GABA-ergic synapse / Stimuli-sensing channels / ruffle membrane / synaptic vesicle / synaptic vesicle membrane / late endosome membrane / late endosome / cytoplasmic vesicle / early endosome membrane / early endosome / endosome membrane / Golgi membrane / lysosomal membrane / external side of plasma membrane / intracellular membrane-bounded organelle / glutamatergic synapse / cell surface / Golgi apparatus / ATP binding / membrane / plasma membrane
Similarity search - Function
Chloride channel ClC-3 / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / Domain in cystathionine beta-synthase and other proteins. / CBS domain superfamily / CBS domain / CBS domain / CBS domain profile.
Similarity search - Domain/homology
CHOLESTEROL / H(+)/Cl(-) exchange transporter 3
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.54 Å
AuthorsSchrecker, M. / Son, Y. / Hite, R.K.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)141553 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)008748 United States
Citation
Journal: Nat Struct Mol Biol / Year: 2025
Title: Structural basis of ClC-3 transporter inhibition by TMEM9 and PtdIns(3,5)P.
Authors: Marina Schrecker / Yeeun Son / Rosa Planells-Cases / Sumanta Kar / Viktoriia Vorobeva / Uwe Schulte / Bernd Fakler / Thomas J Jentsch / Richard K Hite /
Abstract: The trafficking and activity of endosomes relies on the exchange of chloride ions and protons by members of the CLC family of chloride channels and transporters; mutations of the genes encoding these ...The trafficking and activity of endosomes relies on the exchange of chloride ions and protons by members of the CLC family of chloride channels and transporters; mutations of the genes encoding these transporters are associated with numerous diseases. Despite their critical roles, the mechanisms by which CLC transporters are regulated are poorly understood. Here we show that two related accessory β-subunits, TMEM9 and TMEM9B, directly interact with ClC-3, ClC-4 and ClC-5. Cryo-electron microscopy structures reveal that TMEM9 inhibits ClC-3 by sealing the cytosolic entrance to the Cl ion pathway. Unexpectedly, we find that phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P) stabilizes the interaction between TMEM9 and ClC-3 and is required for proper regulation of ClC-3 by TMEM9. Collectively, our findings reveal that TMEM9 and PtdIns(3,5)P collaborate to regulate endosomal ion homeostasis by modulating the activity of ClC-3.
#1: Journal: bioRxiv / Year: 2025
Title: Structural basis of ClC-3 inhibition by TMEM9 and PI(3,5)P.
Authors: Marina Schrecker / Yeeun Son / Rosa Planells-Cases / Sumanta Kar / Viktoriia Vorobeva / Uwe Schulte / Bernd Fakler / Thomas J Jentsch / Richard K Hite /
Abstract: The trafficking and activity of endosomes relies on the exchange of chloride ions and protons by members of the CLC family of chloride channels and transporters, whose mutations are associated with ...The trafficking and activity of endosomes relies on the exchange of chloride ions and protons by members of the CLC family of chloride channels and transporters, whose mutations are associated with numerous diseases. Despite their critical roles, the mechanisms by which CLC transporters are regulated are poorly understood. Here, we show that two related accessory β-subunits, TMEM9 and TMEM9B, directly interact with ClC-3, -4 and -5. Cryo-EM structures reveal that TMEM9 inhibits ClC-3 by sealing the cytosolic entrance to the Cl ion pathway. Unexpectedly, we find that PI(3,5)P stabilizes the interaction between TMEM9 and ClC-3 and is required for proper regulation of ClC-3 by TMEM9. Collectively, our findings reveal that TMEM9 and PI(3,5)P collaborate to regulate endosomal ion homeostasis by modulating the activity of ClC-3.
History
DepositionSep 18, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 2, 2025Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Apr 2, 2025Data content type: Mask / Data content type: Mask / Provider: repository / Type: Initial release
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Revision 1.1May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name
Revision 1.2Sep 24, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin / Item: _em_admin.last_update
Revision 1.3Oct 29, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: H(+)/Cl(-) exchange transporter 3
B: H(+)/Cl(-) exchange transporter 3
hetero molecules


Theoretical massNumber of molelcules
Total (without water)183,79810
Polymers182,1102
Non-polymers1,6888
Water1,874104
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 3 / Chloride channel protein 3 / ClC-3 / Chloride transporter ClC-3


Mass: 91054.977 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLCN3 / Production host: Homo sapiens (human) / References: UniProt: P51790
#2: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-CLR / CHOLESTEROL


Mass: 386.654 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C27H46O / Feature type: SUBJECT OF INVESTIGATION
#4: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 104 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY
Has protein modificationY

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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: Human ClC-3 / 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: 8
Details: 0.02% GDN, 50 mM Tris-HCl (pH 8), 150 mM KCl, 2 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
10.02 %glyco-diosgenin1
250 mMTris1
3150 mMPotassium ChlorideKCl1
42 mMdithiothreitol1
SpecimenConc.: 3 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: 100 % / Chamber temperature: 297 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: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 700 nm
Image recordingAverage exposure time: 3 sec. / Electron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
9PHENIXmodel refinement
10cryoSPARCinitial Euler assignment
11cryoSPARCfinal Euler assignment
12cryoSPARCclassification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1446105
3D reconstructionResolution: 2.54 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 219662 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL / Target criteria: FSC 0.5
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00211314
ELECTRON MICROSCOPYf_angle_d0.41315384
ELECTRON MICROSCOPYf_dihedral_angle_d2.9421540
ELECTRON MICROSCOPYf_chiral_restr0.0351752
ELECTRON MICROSCOPYf_plane_restr0.0041878

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