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- EMDB-41130: Cryo-EM structure of the human CLC-2 chloride channel C-terminal ... -

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Basic information

Entry
Database: EMDB / ID: EMD-41130
TitleCryo-EM structure of the human CLC-2 chloride channel C-terminal domain
Map data
Sample
  • Complex: Chloride channel protein 2
    • Protein or peptide: Chloride channel protein 2
KeywordsChloride / Channel / Inhibitor / Protein / Voltage gated / TRANSPORT PROTEIN
Function / homology
Function and homology information


regulation of aldosterone biosynthetic process / cell differentiation involved in salivary gland development / regulation of membrane depolarization during action potential / volume-sensitive chloride channel activity / stabilization of membrane potential / astrocyte end-foot / acinar cell differentiation / cellular hypotonic response / regulation of resting membrane potential / voltage-gated chloride channel activity ...regulation of aldosterone biosynthetic process / cell differentiation involved in salivary gland development / regulation of membrane depolarization during action potential / volume-sensitive chloride channel activity / stabilization of membrane potential / astrocyte end-foot / acinar cell differentiation / cellular hypotonic response / regulation of resting membrane potential / voltage-gated chloride channel activity / axon initial segment / dendritic spine membrane / chloride channel regulator activity / 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 / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.03 Å
AuthorsXu M / Neelands T / Powers AS / Liu Y / Miller S / Pintilie G / Du Bois J / Dror RO / Chiu W / Maduke M
Funding support United States, 4 items
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, 2023-
Header (metadata) releaseJan 31, 2024-
Map releaseJan 31, 2024-
UpdateFeb 28, 2024-
Current statusFeb 28, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41130.png

Downloads & links

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Map

FileDownload / File: emd_41130.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.95 Å/pix.
x 256 pix.
= 242.176 Å		0.95 Å/pix.
x 256 pix.
= 242.176 Å		0.95 Å/pix.
x 256 pix.
= 242.176 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.946 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.4
Minimum - Maximum-2.563535 - 3.3698559
Average (Standard dev.)0.00008406441 (±0.05057388)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 242.176 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_41130_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41130_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Chloride channel protein 2

EntireName: Chloride channel protein 2
Components
  • Complex: Chloride channel protein 2
    • Protein or peptide: Chloride channel protein 2

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Supramolecule #1: Chloride channel protein 2

SupramoleculeName: Chloride channel protein 2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Chloride channel protein 2

MacromoleculeName: Chloride channel protein 2 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 98.642352 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MAAAAAEEGM EPRALQYEQT LMYGRYTQDL GAFAKEEAAR IRLGGPEPWK GPPSSRAAPE LLEYGRSRCA RCRVCSVRCH KFLVSRVGE DWIFLVLLGL LMALVSWVMD YAIAACLQAQ QWMSRGLNTS ILLQYLAWVT YPVVLITFSA GFTQILAPQA V GSGIPEMK ...String:
MAAAAAEEGM EPRALQYEQT LMYGRYTQDL GAFAKEEAAR IRLGGPEPWK GPPSSRAAPE LLEYGRSRCA RCRVCSVRCH KFLVSRVGE DWIFLVLLGL LMALVSWVMD YAIAACLQAQ QWMSRGLNTS ILLQYLAWVT YPVVLITFSA GFTQILAPQA V GSGIPEMK TILRGVVLKE YLTLKTFIAK VIGLTCALGS GMPLGKEGPF VHIASMCAAL LSKFLSLFGG IYENESRNTE ML AAACAVG VGCCFAAPIG GVLFSIEVTS TFFAVRNYWR GFFAATFSAF IFRVLAVWNR DEETITALFK TRFRLDFPFD LQE LPAFAV IGIASGFGGA LFVYLNRKIV QVMRKQKTIN RFLMRKRLLF PALVTLLIST LTFPPGFGQF MAGQLSQKET LVTL FDNRT WVRQGLVEEL EPPSTSQAWN PPRANVFLTL VIFILMKFWM SALATTIPVP CGAFMPVFVI GAAFGRLVGE SMAAW FPDG IHTDSSTYRI VPGGYAVVGA AALAGAVTHT VSTAVIVFEL TGQIAHILPV MIAVILANAV AQSLQPSLYD SIIRIK KLP YLPELGWGRH QQYRVRVEDI MVRDVPHVAL SCTFRDLRLA LHRTKGRMLA LVESPESMIL LGSIERSQVV ALLGAQL SP ARRRQHMQER RATQTSPLSD QEGPPTPEAS VCFQVNTEDS AFPAARGETH KPLKPALKRG PSVTRNLGES PTGSAESA G IALRSLFCGS PPPEAASEKL ESCEKRKLKR VRISLASDAD LEGEMSPEEI LEWEEQQLDE PVNFSDCKID PAPFQLVER TSLHKTHTIF SLLGVDHAYV TSIGRLIGIV TLKELRKAIE GSVTAQGVKV RPPLASFRDS ATSSSDTETT EVHALWGPHS RHGLPREGS PSDSDDKCQ

UniProtKB: Chloride channel protein 2

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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

BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: COPPER
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Number real images: 14300 / Average exposure time: 5.6 sec. / Average electron dose: 50.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 5214695
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2) / Number images used: 66251
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.2)
Final 3D classificationNumber classes: 10 / Software - Name: cryoSPARC (ver. 3.2)

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Atomic model buiding 1

Initial modelPDB ID:

6qvc


Chain - Source name: SwissModel / Chain - Initial model type: in silico model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT / Target criteria: Q-score
Output model

PDB-8ta6:
Cryo-EM structure of the human CLC-2 chloride channel C-terminal domain

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