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- PDB-6y5r: Structure of Human Potassium Chloride Transporter KCC3 S45D/T940D... -

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Basic information

Entry
Database: PDB / ID: 6y5r
TitleStructure of Human Potassium Chloride Transporter KCC3 S45D/T940D/T997D in NaCl
ComponentsSolute carrier family 12 member 6
KeywordsMEMBRANE PROTEIN / Dimer / transporter / potassium chloride / KCC3 / SLC12A6 / APC / Structural Genomics / Structural Genomics Consortium / SGC / PSI-2 / Protein Structure Initiative
Function / homology
Function and homology information


Defective SLC12A6 causes agenesis of the corpus callosum, with peripheral neuropathy (ACCPN) / potassium ion transmembrane transporter activity / potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / chloride ion homeostasis / cellular hypotonic response / cellular hypotonic salinity response / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane ...Defective SLC12A6 causes agenesis of the corpus callosum, with peripheral neuropathy (ACCPN) / potassium ion transmembrane transporter activity / potassium:chloride symporter activity / Cation-coupled Chloride cotransporters / chloride ion homeostasis / cellular hypotonic response / cellular hypotonic salinity response / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / monoatomic ion transport / chloride transmembrane transport / potassium ion transmembrane transport / cellular response to glucose stimulus / basolateral plasma membrane / chemical synaptic transmission / angiogenesis / axon / synapse / protein kinase binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
K/Cl co-transporter / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / SLC12A transporter family / Amino acid permease
Similarity search - Domain/homology
Solute carrier family 12 member 6
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.76 Å
AuthorsChi, G. / Man, H. / Ebenhoch, R. / Reggiano, G. / Pike, A.C.W. / Wang, D. / McKinley, G. / Mukhopadhyay, S.M.M. / MacLean, B. / Chalk, R. ...Chi, G. / Man, H. / Ebenhoch, R. / Reggiano, G. / Pike, A.C.W. / Wang, D. / McKinley, G. / Mukhopadhyay, S.M.M. / MacLean, B. / Chalk, R. / Moreau, C. / Snee, M. / Bohstedt, T. / Singh, N.K. / Abrusci, P. / Arrowsmith, C.H. / Bountra, C. / Edwards, A.M. / Marsden, B.D. / Burgess-Brown, N.A. / DiMaio, F. / Duerr, K.L. / Structural Genomics Consortium (SGC)
Funding support United Kingdom, 1items
OrganizationGrant numberCountry
European Commission115766 United Kingdom
CitationJournal: To be published
Title: Structure of Human Potassium Chloride Transporter KCC3 in NaCl
Authors: Chi, G. / Man, H. / Ebenhoch, R. / Reggiano, G. / Pike, A.C.W. / Wang, D. / McKinley, G. / Mukhopadhyay, S.M.M. / Chalk, R. / Moreau, C. / Snee, M. / Bohstedt, T. / Singh, N.K. / Abrusci, P. ...Authors: Chi, G. / Man, H. / Ebenhoch, R. / Reggiano, G. / Pike, A.C.W. / Wang, D. / McKinley, G. / Mukhopadhyay, S.M.M. / Chalk, R. / Moreau, C. / Snee, M. / Bohstedt, T. / Singh, N.K. / Abrusci, P. / Arrowsmith, C.H. / Bountra, C. / Edwards, A.M. / Marsden, B.D. / Burgess-Brown, N.A. / DiMaio, F. / Duerr, K.L. / Structural Genomics Consortium (SGC)
History
DepositionFeb 25, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Mar 11, 2020Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Atomic model / Data collection ...Atomic model / Data collection / Derived calculations / Structure summary
Category: atom_site / chem_comp ...atom_site / chem_comp / entity / pdbx_branch_scheme / pdbx_chem_comp_identifier / pdbx_entity_branch / pdbx_entity_branch_descriptor / pdbx_entity_branch_link / pdbx_entity_branch_list / pdbx_entity_nonpoly / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / struct_asym / struct_conn / struct_site / struct_site_gen
Item: _atom_site.B_iso_or_equiv / _atom_site.Cartn_x ..._atom_site.B_iso_or_equiv / _atom_site.Cartn_x / _atom_site.Cartn_y / _atom_site.Cartn_z / _atom_site.auth_asym_id / _atom_site.auth_atom_id / _atom_site.auth_comp_id / _atom_site.auth_seq_id / _atom_site.label_asym_id / _atom_site.label_atom_id / _atom_site.label_comp_id / _atom_site.label_entity_id / _atom_site.type_symbol / _chem_comp.name / _entity.formula_weight / _entity.pdbx_description / _entity.pdbx_number_of_molecules / _entity.type / _pdbx_struct_assembly_gen.asym_id_list / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation
Revision 2.1Nov 13, 2024Group: Data collection / Database references / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_admin / pdbx_entry_details / pdbx_modification_feature
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_admin.last_update / _pdbx_entry_details.has_protein_modification

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Structure visualization

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Structure viewerMolecule:
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Assembly

Deposited unit
A: Solute carrier family 12 member 6
B: Solute carrier family 12 member 6
hetero molecules


Theoretical massNumber of molelcules
Total (without water)246,1626
Polymers244,5472
Non-polymers1,6164
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, homology, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area8860 Å2
ΔGint-12 kcal/mol
Surface area70430 Å2
MethodPISA

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Components

#1: Protein Solute carrier family 12 member 6 / Electroneutral potassium-chloride cotransporter 3 / K-Cl cotransporter 3


Mass: 122273.344 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Sugar molecules from glycosylation post-translational modifications (NAG, BMA) present.
Source: (gene. exp.) Homo sapiens (human) / Gene: SLC12A6, KCC3 / Cell line (production host): HEK293F / Production host: Homo sapiens (human) / References: UniProt: Q9UHW9
#2: Polysaccharide beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta- ...beta-D-mannopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 586.542 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DManpb1-4DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
WURCS=2.0/2,3,2/[a2122h-1b_1-5_2*NCC/3=O][a1122h-1b_1-5]/1-1-2/a4-b1_b4-c1WURCSPDB2Glycan 1.1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-Manp]{}}}}LINUCSPDB-CARE
#3: 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.: 2
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
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
Has ligand of interestN
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: Homodimeric complex of human potassium chloride transporter
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.246 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
SpecimenConc.: 6.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: This sample was monodisperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 297 K
Details: 2 second blotting time, 40 second waiting time, -15 blotting force

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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: -2300 nm / Nominal defocus min: -800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: OTHER
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (max): 70 K / Temperature (min): 70 K
Image recordingElectron dose: 40 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 16472

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Processing

Software
NameVersionClassification
phenix.real_space_refine1.17.1_3660refinement
PHENIX1.17.1_3660refinement
EM software
IDNameVersionCategory
1cisTEM1.0.0bparticle selection
2RELION3.0.8image acquisition
3cisTEM1.0.0bimage acquisition
5Gctf1.16CTF correction
11RELION3.0.8initial Euler assignment
12RELION3.0.8final Euler assignment
13RELION3.0.8classification
14RELION3.0.83D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.76 Å / Resolution method: FSC 0.5 CUT-OFF / Num. of particles: 920574 / Symmetry type: POINT
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 60.58 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.003213895
ELECTRON MICROSCOPYf_angle_d0.537618908
ELECTRON MICROSCOPYf_chiral_restr0.04072252
ELECTRON MICROSCOPYf_plane_restr0.00362348
ELECTRON MICROSCOPYf_dihedral_angle_d6.17791945

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