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- PDB-6ukn: Cryo-EM structure of the potassium-chloride cotransporter KCC4 in... -

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Basic information

Entry
Database: PDB / ID: 6ukn
TitleCryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs
ComponentsSolute carrier family 12 member 7
KeywordsTRANSPORT PROTEIN / Potassium chloride cotransporter / SLC12
Function / homology
Function and homology information


Cation-coupled Chloride cotransporters / ammonium import across plasma membrane / potassium:chloride symporter activity / chloride ion homeostasis / ammonium channel activity / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / plasma membrane => GO:0005886 / chloride transmembrane transport ...Cation-coupled Chloride cotransporters / ammonium import across plasma membrane / potassium:chloride symporter activity / chloride ion homeostasis / ammonium channel activity / potassium ion homeostasis / cell volume homeostasis / potassium ion import across plasma membrane / plasma membrane => GO:0005886 / chloride transmembrane transport / chemical synaptic transmission / synapse / protein kinase binding / protein-containing complex
Similarity search - Function
K/Cl co-transporter 4 / K/Cl co-transporter / SLC12A transporter family / SLC12A transporter, C-terminal / Solute carrier family 12 / Amino acid permease/ SLC12A domain / Amino acid permease
Similarity search - Domain/homology
: / Solute carrier family 12 member 7
Similarity search - Component
Biological speciesMus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.65 Å
AuthorsReid, M.S. / Kern, D.M. / Brohawn, S.G.
Funding support United States, 2items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM123496 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM128263 United States
CitationJournal: Elife / Year: 2020
Title: Cryo-EM structure of the potassium-chloride cotransporter KCC4 in lipid nanodiscs.
Authors: Michelle S Reid / David M Kern / Stephen Graf Brohawn /
Abstract: Cation-chloride-cotransporters (CCCs) catalyze transport of Cl with K and/or Naacross cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, ...Cation-chloride-cotransporters (CCCs) catalyze transport of Cl with K and/or Naacross cellular membranes. CCCs play roles in cellular volume regulation, neural development and function, audition, regulation of blood pressure, and renal function. CCCs are targets of clinically important drugs including loop diuretics and their disruption has been implicated in pathophysiology including epilepsy, hearing loss, and the genetic disorders Andermann, Gitelman, and Bartter syndromes. Here we present the structure of a CCC, the K-Cl cotransporter (KCC) KCC4, in lipid nanodiscs determined by cryo-EM. The structure, captured in an inside-open conformation, reveals the architecture of KCCs including an extracellular domain poised to regulate transport activity through an outer gate. We identify binding sites for substrate K and Cl ions, demonstrate the importance of key coordinating residues for transporter activity, and provide a structural explanation for varied substrate specificity and ion transport ratio among CCCs. These results provide mechanistic insight into the function and regulation of a physiologically important transporter family.
History
DepositionOct 5, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 2.0Jul 29, 2020Group: Advisory / Atomic model ...Advisory / 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 / pdbx_struct_conn_angle / pdbx_validate_close_contact / struct_asym / struct_conn / struct_conn_type / 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 / _pdbx_struct_assembly_gen.asym_id_list / _pdbx_struct_conn_angle.ptnr2_label_asym_id / _pdbx_validate_close_contact.auth_asym_id_1 / _pdbx_validate_close_contact.auth_asym_id_2 / _pdbx_validate_close_contact.auth_seq_id_1 / _pdbx_validate_close_contact.auth_seq_id_2 / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
A: Solute carrier family 12 member 7
hetero molecules


Theoretical massNumber of molelcules
Total (without water)121,8595
Polymers120,6111
Non-polymers1,2484
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area1550 Å2
ΔGint3 kcal/mol
Surface area25050 Å2

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Components

#1: Protein Solute carrier family 12 member 7 / Electroneutral potassium-chloride cotransporter 4 / K-Cl cotransporter 4


Mass: 120610.961 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Gene: Slc12a7, Kcc4 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q9WVL3
#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: Chemical ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: K / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-CL / CHLORIDE ION / Chloride


Mass: 35.453 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Cl / Feature type: SUBJECT OF INVESTIGATION
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: KCC4 in lipid nanodiscs / Type: CELL / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Mus musculus (house mouse)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris1
2150 mMPotassium ChlorideKCl1
31 mMEDTAEthylenediaminetetraacetic acid1
SpecimenConc.: 1 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 / Details: 3uL, 1 blot force, 4s blot time, 1s wait time

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 36000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 0.11 sec. / Electron dose: 0.9333 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategory
2SerialEMimage acquisition
4CTFFIND4.1.13CTF correction
7Cootmodel fitting
9cryoSPARC2initial Euler assignment
10RELION3.0.7final Euler assignment
11RELION3.0.7classification
12RELION3.0.73D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1826000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.65 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 110143 / Symmetry type: POINT

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