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- EMDB-47068: Human ClC-3:TMEM9, TMEM9 Protomer A: No CDTMEM9, Protomer B: No L... -

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Basic information

Entry
Database: EMDB / ID: EMD-47068
TitleHuman ClC-3:TMEM9, TMEM9 Protomer A: No CDTMEM9, Protomer B: No LD, No CD
Map data
Sample
  • Complex: Human ClC-3 and Human TMEM9
    • Protein or peptide: H(+)/Cl(-) exchange transporter 3
    • Protein or peptide: Proton-transporting V-type ATPase complex assembly regulator TMEM9
  • Ligand: CHLORIDE ION
  • Ligand: CHOLESTEROL
  • Ligand: (2R)-1-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl (5E,8E,11E,13E)-icosa-5,8,11,13-tetraenoate
KeywordsIon channel / membrane protein / lysosomal protein / CLC
Function / homology
Function and homology information


proton-transporting V-type ATPase complex assembly / volume-sensitive chloride channel activity / chloride:proton antiporter activity / synaptic vesicle lumen acidification / negative regulation of cell volume / lysosomal lumen acidification / endosomal lumen acidification / regulation of pH / voltage-gated chloride channel activity / specific granule ...proton-transporting V-type ATPase complex assembly / volume-sensitive chloride channel activity / chloride:proton antiporter activity / synaptic vesicle lumen acidification / negative regulation of cell volume / lysosomal lumen acidification / endosomal lumen acidification / regulation of pH / voltage-gated chloride channel activity / specific granule / multivesicular body membrane / photoreceptor cell maintenance / vesicle membrane / antiporter activity / synaptic transmission, GABAergic / phagocytosis, engulfment / positive regulation of reactive oxygen species biosynthetic process / chloride channel activity / regulation of protein catabolic process / intercellular bridge / phagocytic vesicle / axon terminus / chloride transmembrane transport / secretory granule / adult locomotory behavior / GABA-ergic synapse / synaptic transmission, glutamatergic / PDZ domain binding / recycling endosome / Stimuli-sensing channels / ruffle membrane / synaptic vesicle membrane / mitotic spindle / late endosome membrane / positive regulation of canonical Wnt signaling pathway / late endosome / synaptic vesicle / protein transport / microtubule cytoskeleton / early endosome membrane / cytoplasmic vesicle / early endosome / lysosome / endosome membrane / Golgi membrane / external side of plasma membrane / lysosomal membrane / intracellular membrane-bounded organelle / glutamatergic synapse / cell surface / Golgi apparatus / ATP binding / membrane / plasma membrane
Similarity search - Function
TMEM9/TMEM9B / TMEM9 / 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
H(+)/Cl(-) exchange transporter 3 / Proton-transporting V-type ATPase complex assembly regulator TMEM9
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.01 Å
AuthorsSon Y / Schrecker M / Hite RK
Funding support United States, 2 items
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
CitationJournal: 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, 2024-
Header (metadata) releaseApr 2, 2025-
Map releaseApr 2, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_47068.png

Downloads & links

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Map

FileDownload / File: emd_47068.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.73 Å/pix.
x 384 pix.
= 278.4 Å		0.73 Å/pix.
x 384 pix.
= 278.4 Å		0.73 Å/pix.
x 384 pix.
= 278.4 Å

Surface

Projections

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Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.725 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0329
Minimum - Maximum-0.060366426 - 0.1333768
Average (Standard dev.)0.00016833113 (±0.003707304)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 278.40002 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_47068_msk_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #2

Fileemd_47068_half_map_1.map
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Half map: #1

Fileemd_47068_half_map_2.map
Projections & Slices
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Sample components

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Entire : Human ClC-3 and Human TMEM9

EntireName: Human ClC-3 and Human TMEM9
Components
  • Complex: Human ClC-3 and Human TMEM9
    • Protein or peptide: H(+)/Cl(-) exchange transporter 3
    • Protein or peptide: Proton-transporting V-type ATPase complex assembly regulator TMEM9
  • Ligand: CHLORIDE ION
  • Ligand: CHOLESTEROL
  • Ligand: (2R)-1-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl (5E,8E,11E,13E)-icosa-5,8,11,13-tetraenoate

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Supramolecule #1: Human ClC-3 and Human TMEM9

SupramoleculeName: Human ClC-3 and Human TMEM9 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: H(+)/Cl(-) exchange transporter 3

MacromoleculeName: H(+)/Cl(-) exchange transporter 3 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 91.054977 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MESEQLFHRG YYRNSYNSIT SASSDEELLD GAGVIMDFQT SEDDNLLDGD TAVGTHYTMT NGGSINSSTH LLDLLDEPIP GVGTYDDFH TIDWVREKCK DRERHRRINS KKKESAWEMT KSLYDAWSGW LVVTLTGLAS GALAGLIDIA ADWMTDLKEG I CLSALWYN ...String:
MESEQLFHRG YYRNSYNSIT SASSDEELLD GAGVIMDFQT SEDDNLLDGD TAVGTHYTMT NGGSINSSTH LLDLLDEPIP GVGTYDDFH TIDWVREKCK DRERHRRINS KKKESAWEMT KSLYDAWSGW LVVTLTGLAS GALAGLIDIA ADWMTDLKEG I CLSALWYN HEQCCWGSNE TTFEERDKCP QWKTWAELII GQAEGPGSYI MNYIMYIFWA LSFAFLAVSL VKVFAPYACG SG IPEIKTI LSGFIIRGYL GKWTLMIKTI TLVLAVASGL SLGKEGPLVH VACCCGNIFS YLFPKYSTNE AKKREVLSAA SAA GVSVAF GAPIGGVLFS LEEVSYYFPL KTLWRSFFAA LVAAFVLRSI NPFGNSRLVL FYVEYHTPWY LFELFPFILL GVFG GLWGA FFIRANIAWC RRRKSTKFGK YPVLEVIIVA AITAVIAFPN PYTRLNTSEL IKELFTDCGP LESSSLCDYR NDMNA SKIV DDIPDRPAGI GVYSAIWQLC LALIFKIIMT VFTFGIKVPS GLFIPSMAIG AIAGRIVGIA VEQLAYYHHD WFIFKE WCE VGADCITPGL YAMVGAAACL GGVTRMTVSL VVIVFELTGG LEYIVPLMAA VMTSKWVGDA FGREGIYEAH IRLNGYP FL DAKEEFTHTT LAADVMRPRR NDPPLAVLTQ DNMTVDDIEN MINETSYNGF PVIMSKESQR LVGFALRRDL TIAIESAR K KQEGIVGSSR VCFAQHTPSL PAESPRPLKL RSILDMSPFT VTDHTPMEIV VDIFRKLGLR QCLVTHNGRL LGIITKKDI LRHMAQTANQ DPASIMFN

UniProtKB: H(+)/Cl(-) exchange transporter 3

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Macromolecule #2: Proton-transporting V-type ATPase complex assembly regulator TMEM9

MacromoleculeName: Proton-transporting V-type ATPase complex assembly regulator TMEM9
type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 20.598934 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
MKLLSLVAVV GCLLVPPAEA NKSSEDIRCK CICPPYRNIS GHIYNQNVSQ KDCNCLHVVE PMPVPGHDVE AYCLLCECRY EERSTTTIK VIIVIYLSVV GALLLYMAFL MLVDPLIRKP DAYTEQLHNE EENEDARSMA AAAASLGGPR ANTVLERVEG A QQRWKLQV QEQRKTVFDR HKMLS

UniProtKB: Proton-transporting V-type ATPase complex assembly regulator TMEM9

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Macromolecule #3: CHLORIDE ION

MacromoleculeName: CHLORIDE ION / type: ligand / ID: 3 / Number of copies: 4 / Formula: CL
Molecular weightTheoretical: 35.453 Da

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Macromolecule #4: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 4 / Number of copies: 4 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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Macromolecule #5: (2R)-1-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bi...

MacromoleculeName: (2R)-1-{[(S)-hydroxy{[(1S,2R,3R,4S,5S,6R)-2,4,6-trihydroxy-3,5-bis(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}-3-(octadecanoyloxy)propan-2-yl (5E,8E,11E,13E)-icosa-5,8,11,13-tetraenoate
type: ligand / ID: 5 / Number of copies: 2 / Formula: A1A8I
Molecular weightTheoretical: 1.047088 KDa

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration5 mg/mL
BufferpH: 8
Component:
ConcentrationNameFormula
0.02 %glyco-diosgenin
50.0 mMTris
150.0 mMPotassium ChlorideKCl
2.0 mMdithiothreitol

Details: 0.02% GDN, 50 mM Tris-HCl (pH 8), 150 mM KCl, 2 mM DTT
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 297 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average exposure time: 2.93 sec. / Average electron dose: 59.63 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 10137040
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER / Details: cryosparc ab-initio
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.01 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 91755
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: BACKBONE TRACE / Target criteria: FSC 0.5
Output model

PDB-9dny:
Human ClC-3:TMEM9, TMEM9 Protomer A: No CD TMEM9, Protomer B: No LD, No CD

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