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- PDB-7jm7: Structure of human CLC-7/OSTM1 complex -

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Basic information

Entry
Database: PDB / ID: 7jm7
TitleStructure of human CLC-7/OSTM1 complex
Components
  • H(+)/Cl(-) exchange transporter 7
  • Osteopetrosis-associated transmembrane protein 1
KeywordsMEMBRANE PROTEIN / Lysosomal / chloride-proton antiporter / chloride transport / ion transport / proton transport / complex / glycosylated
Function / homology
Function and homology information


transepithelial chloride transport / voltage-gated chloride channel activity / antiporter activity / chloride transmembrane transporter activity / chloride channel activity / chloride channel complex / osteoclast differentiation / Stimuli-sensing channels / lysosomal membrane / intracellular membrane-bounded organelle ...transepithelial chloride transport / voltage-gated chloride channel activity / antiporter activity / chloride transmembrane transporter activity / chloride channel activity / chloride channel complex / osteoclast differentiation / Stimuli-sensing channels / lysosomal membrane / intracellular membrane-bounded organelle / ATP binding / membrane / cytosol
Similarity search - Function
Osteopetrosis-associated transmembrane protein 1 precursor / Osteopetrosis-associated transmembrane protein 1 precursor / Chloride channel ClC-7 / Chloride channel, voltage gated / Chloride channel, core / Voltage gated chloride channel / CBS domain superfamily / Domain in cystathionine beta-synthase and other proteins. / CBS domain / CBS domain / CBS domain profile.
Similarity search - Domain/homology
Chem-0J1 / ADENOSINE-5'-TRIPHOSPHATE / H(+)/Cl(-) exchange transporter 7 / Osteopetrosis-associated transmembrane protein 1
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.82 Å
AuthorsSchrecker, M. / Hite, R.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)P30 CA008748 United States
CitationJournal: Elife / Year: 2020
Title: Cryo-EM structure of the lysosomal chloride-proton exchanger CLC-7 in complex with OSTM1.
Authors: Marina Schrecker / Julia Korobenko / Richard K Hite /
Abstract: The chloride-proton exchanger CLC-7 plays critical roles in lysosomal homeostasis and bone regeneration and its mutation can lead to osteopetrosis, lysosomal storage disease and neurological ...The chloride-proton exchanger CLC-7 plays critical roles in lysosomal homeostasis and bone regeneration and its mutation can lead to osteopetrosis, lysosomal storage disease and neurological disorders. In lysosomes and the ruffled border of osteoclasts, CLC-7 requires a β-subunit, OSTM1, for stability and activity. Here, we present electron cryomicroscopy structures of CLC-7 in occluded states by itself and in complex with OSTM1, determined at resolutions up to 2.8 Å. In the complex, the luminal surface of CLC-7 is entirely covered by a dimer of the heavily glycosylated and disulfide-bonded OSTM1, which serves to protect CLC-7 from the degradative environment of the lysosomal lumen. OSTM1 binding does not induce large-scale rearrangements of CLC-7, but does have minor effects on the conformation of the ion-conduction pathway, potentially contributing to its regulatory role. These studies provide insights into the role of OSTM1 and serve as a foundation for understanding the mechanisms of CLC-7 regulation.
History
DepositionJul 31, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 2, 2020Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
B: Osteopetrosis-associated transmembrane protein 1
A: H(+)/Cl(-) exchange transporter 7
D: Osteopetrosis-associated transmembrane protein 1
C: H(+)/Cl(-) exchange transporter 7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)260,08330
Polymers252,1294
Non-polymers7,95426
Water64936
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Noncrystallographic symmetry (NCS)NCS domain:
IDEns-IDDetails
d_1ens_1chain "A"
d_2ens_1chain "C"
d_1ens_2chain "D"
d_2ens_2chain "B"

NCS ensembles :
ID
ens_1
ens_2

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Components

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Protein , 2 types, 4 molecules BDAC

#1: Protein Osteopetrosis-associated transmembrane protein 1 / Chloride channel 7 beta subunit


Mass: 37290.570 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: OSTM1, GL, HSPC019, UNQ6098/PRO21201 / Production host: Homo sapiens (human) / References: UniProt: Q86WC4
#2: Protein H(+)/Cl(-) exchange transporter 7 / Chloride channel 7 alpha subunit / Chloride channel protein 7 / ClC-7


Mass: 88773.875 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: CLCN7 / Production host: Homo sapiens (human) / References: UniProt: P51798

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Sugars , 3 types, 14 molecules

#3: Polysaccharide alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2- ...alpha-D-mannopyranose-(1-3)-[alpha-D-mannopyranose-(1-6)]beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 910.823 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpa1-3[DManpa1-6]DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/3,5,4/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5][a1122h-1a_1-5]/1-1-2-3-3/a4-b1_b4-c1_c3-d1_c6-e1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{[(3+1)][a-D-Manp]{}[(6+1)][a-D-Manp]{}}}}LINUCSPDB-CARE
#4: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-ROHGlycam Condensed SequenceGMML 1.0
WURCS=2.0/1,2,1/[a2122h-1b_1-5_2*NCC/3=O]/1-1/a4-b1WURCSPDB2Glycan 1.1.0
[][D-1-deoxy-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}LINUCSPDB-CARE
#5: Sugar
ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-acetyl-beta-D-glucosamine / 2-acetamido-2-deoxy-beta-D-glucose / 2-acetamido-2-deoxy-D-glucose / 2-acetamido-2-deoxy-glucose / N-ACETYL-D-GLUCOSAMINE


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C8H15NO6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 5 types, 48 molecules

#6: Chemical ChemComp-ATP / ADENOSINE-5'-TRIPHOSPHATE


Mass: 507.181 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C10H16N5O13P3 / Comment: ATP, energy-carrying molecule*YM
#7: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
#8: Chemical
ChemComp-CL / CHLORIDE ION


Mass: 35.453 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Cl
#9: Chemical ChemComp-0J1 / (2R)-3-{[(R)-hydroxy{[(1S,2R,3S,4S,5R,6R)-2,3,4,6-tetrahydroxy-5-(phosphonooxy)cyclohexyl]oxy}phosphoryl]oxy}propane-1,2-diyl dinonanoate


Mass: 694.640 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C27H52O16P2 / Feature type: SUBJECT OF INVESTIGATION
#10: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 36 / Source method: isolated from a natural source / Formula: H2O

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Details

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: Human CLC-7/OSTM1 complex / Type: COMPLEX / Entity ID: #1-#2 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Homo (humans)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 8
Buffer component
IDConc.FormulaBuffer-ID
1150 mMKCL1
250 mMTris1
32 mMDTT1
40.1 %LMNG1
51 mMATP1
SpecimenConc.: 2 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: 277 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: OTHER / Nominal magnification: 22500 X / Nominal defocus max: -2700 nm / Nominal defocus min: -1200 nm / Cs: 2.7 mm
Image recordingAverage exposure time: 8 sec. / Electron dose: 44 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k)

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.18_3861refinement
PHENIX1.18_3861refinement
EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFINDCTF correction
9PHENIXmodel refinement
11cryoSPARCV2.15.0final Euler assignment
12cryoSPARCV2.15.0classification
13cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.82 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 327619 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 48.22 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003914810
ELECTRON MICROSCOPYf_angle_d0.711220124
ELECTRON MICROSCOPYf_chiral_restr0.11792412
ELECTRON MICROSCOPYf_plane_restr0.00462474
ELECTRON MICROSCOPYf_dihedral_angle_d20.03075360

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