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Horizontal Tabs
PDB ID |
Structure Details |
WNK1 CCT2 domain |
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WNK2 CCT1 domain |
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WNK2 CCT1 domain + RFXV-containing peptide |
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WNK3 degron peptide + KLHL3 kelch domain |
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WNK4 degron peptide + KLHL2 kelch domain |
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WNK4 degron peptide + KLHL3 kelch domain |
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WNK3 kinase monophosphorylated apo |
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WNK3 kinase A-loop exchange |
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WNK3 kinase + inhibitory Cl- ion |
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WNK3 kinase diphosphorylated + AMP-PNP |
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WNK3 kinase + PP121 inhibitor |
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WNK3 kinase-CCT1 fusion |
Thermal Shift (DSF) Assay
A fluorescence-based thermal shift assay (DSF) was performed as a screen to identify potential WNK3 inhibitors. Ligands in this assay increase a protein’s melting temperature (Tm shift) by an amount proportional to their binding affinity. A solution of 1-2 mM WNK3-CCT1 protein in assay buffer (10 mM HEPES pH 7.5, 150 mM NaCl) was mixed 1:1000 with SYPRO Orange (Invitrogen). Compounds to be tested were added to a final concentration of 12.5 mM. 20 mL of each sample were placed in a 96-well plate and heated from 25 to 95°C. Fluorescence was monitored using a Mx3005P real-time PCR instrument (Agilent) with excitation and emission filters set to 465 and 590 nm, respectively. Data were analysed with the MxPro software and curves fit in Microsoft Excel using the Boltzmann equation to determine the midpoint of thermal denaturation (Tm). Thermal shift values (DTm) induced by inhibitor binding were calculated relative to control wells containing protein and 2.5% DMSO.
In vitro Kinase Assay
General protocol: Test samples in 96 well plates are set up containing 0.5 mL inhibitor, 15 mL enzyme, substrate and buffer and incubated at room temperature for 5 min. The assay is initiated by the addition of 10 mL 33P Mg-ATP (at approximately Km for ATP) and incubated at room temperature for 30 min. The assay is then stopped by the addition of orthophosphoric acid, harvested onto P81 filterplates, dried and read on a scintillation counter for 30 sec per well. The assays are published in Biochem J. (2003) 371, 199-204 and Biochem J (2007) 408, 297-315.
WNK3 specific protocol: WNK3 (5-20 mU diluted in 20 mM MOPS pH 7, 1 mM EGTA, 0.01% Brij35, 5% glycerol, 1 mg/mL BSA, 0.1% mercaptoethanol) is assayed against myelin basic protein, MBP, in a final volume of 25.5 µL containing 8 mM MOPS pH 7, 0.02 mM EDTA, 5 mM MnCl2, 0.33 mg/mL MBP, 10 mM magnesium acetate and 0.005 mM [33P-γ-ATP] (50-1000 cpm/pmole) and incubated for 30 min at room temperature. Assays are stopped by addition of 5 µL of 0.5 M (3%) orthophosphoric acid and then harvested onto P81 Unifilter plates with a wash buffer of 50 mM orthophosphoric acid.
Mass spectrometry
Protein masses were determined using an Agilent LC/MSD TOF system with reversed-phase high-performance liquid chromatography coupled to electrospray ionization and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95% isopropanol in water with 0.1% formic acid. Spectra were analysed using the MassHunter software (Agilent).
Fluorescence polarisation assay for WNK degron binding to KLHL2/3 kelch domains
Fluorescence polarisation measurements were performed at 25°C with purified KLHL3 proteins in 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT. All peptides contained an N-terminal linker required for conjugating to the Lumio green flurophore (CCPGCCGGGG) and were initially resuspended in 50 mM ammonium biocarbonate pH 8. Peptide labelling was achieved by incubating 10 nM of each peptide in a 0.5 mL reaction mixture of 20 μM Lumio green in 50 mM TrisHCl pH 7.5, 150 mM NaCl, 2 mM DTT. Reactions were left to proceed in the dark for 2 h. The peptides were then dialysed for 16 h into 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM DTT using a Micro DispoDIALYZER with a 100 Da molecular weight cut off (Harvard Apparatus). For fluorescence polarisation, mixtures were set up containing the indicated concentration of protein, 10 nM Lumio-green labelled peptide in a final volume of 30 mL. All individual bindings were performed in duplicate with at least 12 data points per curve. Fluorescence polarisation measures were made using a BMG PheraStar plate reader, with an excitation wavelength of 485 nm and an emission wavelength of 538 nm, and measurements were corrected to the fluorescent probe alone. Data analysis and graphing were then performed in GraphPad Prism; One Site Specific binding with Hill Slope was assumed (model Y=Bmax*X^h/Kd^h + X^h) and the disassociation constant, and associated standard error were obtained. All experimental bindings were repeated a minimum of two times and comparable results were obtained.
Preparation of Human KLHL3 kelch domain
Boundaries: residues 298–587
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
Human KLHL3 kelch domain was expressed from the vector pNIC28-Bsa4 in BL21(DE3)-R3-pRARE cells (a phage-resistant derivative of Rosetta2, Novagen). Cultures (1 litre) in Terrific broth were incubated at 37°C until OD600 reached 2.0 and then cooled to 18°C and supplemented with 0.5 mM IPTG to induce protein expression overnight. Cells were harvested by centrifugation, resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol and 0.5 mM TCEP [tris-(2-carboxyethyl)phosphine] and lysed by sonication. The His6-tagged protein was purified using Ni2+-Sepharose resin (GE Healthcare) and eluted stepwise in binding buffer with 100-250 mM imidazole. Removal of the His6 tag was performed at 4°C overnight using TEV protease. KLHL3 was then further purified by anion exchange chromatography using a 5 mL HiTrap Q column (GE Healthcare) followed by a final buffer-exchange step into 50 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol and 0.5 mM TCEP using size-exclusion chromatography (Superdex 200 16/60, GE Healthcare).
Preparation of Human KLHL2 kelch domain
Boundaries: residues 294–593
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
Human KLHL2 kelch domain was expressed from the vector pNIC28-Bsa4 in BL21(DE3)-R3-pRARE cells (a phage-resistant derivative of Rosetta2, Novagen). Cultures (1 litre) in Terrific broth were incubated at 37°C until OD600 reached 2.0 and then cooled to 18°C and supplemented with 0.5 mM IPTG to induce protein expression overnight. Cells were harvested by centrifugation, resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5 mM imidazole, 5% glycerol and 0.5 mM TCEP [tris-(2-carboxyethyl)phosphine] and lysed by sonication. The His6-tagged protein was purified using Ni2+-Sepharose resin (GE Healthcare) and eluted stepwise in binding
buffer with 100-250 mM imidazole. Removal of the His6 tag was performed at 4°C overnight using TEV protease. KLHL2 was then further purified by cation exchange chromatography using a 5 mL HiTrap SP column (GE Healthcare) followed by a buffer-exchange step into 50 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol and 0.5 mM TCEP using size-exclusion chromatography (Superdex 200 16/60, GE Healthcare).
Preparation of Human WNK3 kinase domain (diphosphorylated)
Boundaries: residues 133–414
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
Human WNK3 kinase domain was expressed from the vector pNIC28-Bsa4 in BL21(DE3)-R3-pRARE cells. Cultures were grown at 37°C in LB medium supplemented with 50 μg/mL kanamycin and 34 µg/mL chloramphenicol to an OD600 of 0.6, before expression at 18°C overnight and induction with 0.3 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). Cells were then harvested by centrifugation at 5000g and resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole, EDTA-free complete protease inhibitor cocktail (Roche)) and lysed by sonication. Lysates were clarified by centrifugation in a JA 25.50 rotor at 36000 g. His-tagged protein was immobilized on Ni2+-sepharose resin and bound proteins eluted using step gradients of Imidazole (50-250 mM). The eluted proteins were cleaved with TEV protease overnight at 4°C and further purified by size exclusion chromatography using an S200 HiLoad 16/60 Superdex column equilibrated in buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl, and 0.5 mM TCEP. Proteins were concentrated using centrifugal ultrafiltration with a 10 kDa molecular weight cut-off point membrane. Protein purity of >95% was confirmed by SDS-PAGE and identity plus phosphorylation state verified by intact mass spectrometry. Bacterial expression yielded diphosphorylated WNK3 protein (Ser304 and Ser308). When desired the non-phosphorylated kinase domain was prepared by in vitro lambda phosphatase treatment or by bacterial coexpression with lambda phosphatase.
Preparation of Human WNK3 kinase domain (monophosphorylated)
Boundaries: residues 132–414
Vector: pFB-LIC-Bse
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: Sf9 insect cells
Bacmid DNA was prepared from DH10Bac cells and used to transfect Sf9 insect cells for the preparation of initial baculovirus. WNK3 protein was expressed from infected Sf9 cells cultivated in InsectXpress medium (Lonza) for 48 hours at 27°C. Harvested cells were resuspended and purified similarly to the bacterially-expressed WNK3 kinase domain described above. Baculoviral expression resulted in predominantly Ser304 monophosphorylated WNK3 protein.
Preparation of Human WNK3 kinase-CCT1 domain fusion
Boundaries: residues 123–500
Vector: pFB-LIC-Bse
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: Sf9 insect cells
Bacmid DNA was prepared from DH10Bac cells and used to transfect Sf9 insect cells for the preparation of initial baculovirus. WNK3 protein was expressed from infected Sf9 cells cultivated in InsectXpress medium (Lonza) for 48 hours at 27°C. Harvested cells were resuspended and purified similarly to the bacterially-expressed WNK3 kinase domain described above. When required dephosphorylated WNK3 was prepared by co-infection of baculovirus expressing lambda phosphatase.
Preparation of Human WNK3 CCT1 domain
Boundaries: residues 406–500
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
See general protocol for preparation of CCT domains below.
Preparation of Human WNK3 CCT2 domain
Boundaries: residues 741–820
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
See general protocol for preparation of CCT domains below.
Preparation of Human WNK1 CCT1 domain
Boundaries: residues 480–572
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
See general protocol for preparation of CCT domains below.
Preparation of Human WNK2 CCT1 domain
Boundaries: residues 454-549
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
See general protocol for preparation of CCT domains below.
Preparation of Human WNK1 CCT2 domain
Boundaries: residues 1115–1194
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
See general protocol for preparation of CCT domains below.
Preparation of Human WNK4 CCT2 domain
Boundaries: residues 671–751
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
See general protocol for preparation of CCT domains below.
Preparation of Human SPAK CCT domain
Boundaries: residues 448–547
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
See general protocol for preparation of CCT domains below.
Preparation of Human OSR1 CCT domain
Boundaries: residues 432–527
Vector: pNIC28-Bsa4
Tag and additions: TEV-cleavable N-terminal hexahistidine tag
Expression cell: E. coli BL21(DE3)R3-pRARE2
See general protocol for preparation of CCT domains below.
General procedure for CCT domain preparation
Human CCT domains were expressed from the vector pNIC28-Bsa4 in BL21(DE3)-R3-pRARE cells. Cultures were grown at 37°C in LB medium supplemented with 50 μg/mL kanamycin and 34 µg/mL chloramphenicol to an OD600 of 0.6, before expression at 18°C overnight and induction with 0.3 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). Cells were then harvested by centrifugation at 5000g and resuspended in binding buffer (50 mM HEPES pH 7.5, 500 mM NaCl, 5% glycerol, 5 mM imidazole, EDTA-free complete protease inhibitor cocktail (Roche)) and lysed by sonication. Lysates were clarified by centrifugation in a JA 25.50 rotor at 36000 g. His-tagged protein was immobilized on Ni2+-sepharose resin and bound proteins eluted using step gradients of Imidazole (50-250 mM). The eluted proteins were cleaved with TEV protease overnight at 4°C and further purified by size exclusion chromatography using an S75 HiLoad 16/60 Superdex column equilibrated in buffer containing 50 mM HEPES pH 7.5, 300 mM NaCl, and 0.5 mM TCEP. Proteins were concentrated using centrifugal ultrafiltration with a 3 kDa molecular weight cut-off point membrane. Protein purity of >95% was confirmed by SDS-PAGE and identity verified by intact mass spectrometry. Protein concentrations were determined by measuring absorbance at 280 nm, or in cases where extinction coefficients were too low, concentrations were determined using Bradford assays and validated with a Direct Detect Infrared Spectrometer.
Structure Determination of the KLHL3/WNK4 complex (PDB: 4CH9)
KLHL3 protein was concentrated to 9 mg/mL and mixed with 2 mM final concentration WNK4 peptide (EPEEPEADQHQ) for 30 minutes on ice. Crystallisation was performed using sitting-drop vapour-diffusion. Crystals of the KLHL3 complex were obtained at 20°C by mixing 100 nL of protein with 50 nL of a reservoir solution containing 0.1 M acetate pH 4.3, 0.2 M ammonium sulphate and 25-35 % PEG 4000. After 3 hours of incubation the drops were spiked with 20 nL of seed-stock solution. Prior to vitrification in liquid nitrogen, crystals were cryoprotected by direct addition of reservoir solution supplemented with 25 % ethylene glycol. Diffraction data were collected on beamline I02 at Diamond Light Source. Diffraction data were integrated using Mosflm and scaled using SCALA from the CCP4 software suite. Molecular replacement was performed with Phaser MR in CCP4 using PDB ID code 2XN4 chain A (apo kelch domain of KLHL2) as the search model. COOT was used for manual model building and refinement, while REFMAC and PHENIX.REFINE were used for automated refinement. TLS parameters were included at later stages of refinement. Tools in COOT, PHENIX and MolProbity were used to validate the structures.
Structure Determination of the KLHL2/WNK4 complex (PDB: 4CHB)
KLHL2 protein was concentrated to 9 mg/mL and mixed with 2 mM final concentration WNK4 peptide (EPEEPEADQHQ). The protein-peptide solution was incubated on ice for approximately 30 minutes prior to preparation of sitting-drop vapour-diffusion crystallisation plates. Diffracting crystals were obtained at 20°C by mixing 100 nL of protein with 50 nL of a reservoir solution containing 0.1 M HEPES pH 7.2, 2.5 M ammonium sulphate and 2 % PEG 400. After 3 hours of incubation the drops were spiked with 20 nL of seed-stock solution. Prior to vitrification in liquid nitrogen, crystals were cryoprotected by direct addition of reservoir solution supplemented with 25 % ethylene glycol. Diffraction data were collected on beamline I02 at Diamond Light Source. Diffraction data were integrated using Mosflm and scaled using SCALA from the CCP4 software suite. Molecular replacement was performed with Phaser MR in CCP4 using PDB ID code 2XN4 chain A (apo kelch domain of KLHL2) as the search model. COOT was used for manual model building and refinement, while REFMAC and PHENIX.REFINE were used for automated refinement. TLS parameters were included at later stages of refinement. Tools in COOT, PHENIX and MolProbity were used to validate the structures.
Structure Determination of the KLHL3/WNK3 complex (PDB: 5NKP)
KLHL3 was concentrated to 9 mg/mL buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP, 2 mM DTT and mixed with 2 mM WNK3 acidic motif peptide (ECEETEVDQHV). Diffracting crystals were obtained at 4°C in sitting drops by mixing 100 nL protein with 50 nL solution containing 6% PEG4K, 0.1 M acetate pH 5.1. After 3 hours of incubation the drops were spiked with 20 nL of seed-stock solution. Prior to vitrification in liquid nitrogen, crystals were cryoprotected by direct addition of reservoir solution supplemented with 25 % ethylene glycol. Diffraction data were collected on beamline I03 at Diamond Light Source. Diffraction data were processed using PHENIX. Molecular replacement was performed with Phaser MR in Phenix using PDB 4CH9 as the model. COOT was used for manual model building and refinement, whereas PHENIX.REFINE was used for automated refinement. TLS parameters were included at later stages of refinement.
General methods for structure determination of WNK kinase and CCT domains
Data were indexed and integrated using XDS and scaled using AIMLESS in the CCP4 suite of programs. Phases were found using molecular replacement in PHASER. Models were built initially using COOT and then refined and modified using alternate rounds of REFMAC5 and COOT. Specific details for each structure are listed below.
Structure determination of WNK3 kinase-CCT1 (dephosphorylated)
Dephosphorylated WNK3 Kinase-CCT1 (a.a. 123-500) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 0.5 mM TCEP, and concentrated to 9.6 mg/mL. Crystals were grown at 4°C in 400 nL sitting drops at a 1:1 protein: reservoir solution comprising 18% PEG3350, 0.1 M citrate pH 5. Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25 % glycerol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I03.
Structure determination of WNK3 kinase domain (apo, monophosphorylated)
Monophosphorylated WNK3 kinase domain (a.a. 132-414) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP, and concentrated to 12.3 mg/mL. Crystals were grown at 20°C in 150 nL sitting drops at a 1:1 protein: reservoir solution comprising 30% PEG Smear Medium, 0.2 M lithium sulfate, 0.1 M ADA pH 6.5. Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25% ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I03.
Structure determination of WNK3 kinase (Cl- bound, monophosphorylated)
Monophosphorylated WNK3 kinase domain (a.a. 132-414) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP, and concentrated to 10 mg/mL. 1 mM adenosine diphosphate (ADP) and 2 mM MgCl2 were added, but not observed in the resulting structure. Crystals were grown at 4°C in 150 nL sitting drops at a 2:1 protein: reservoir solution comprising 22.5% PEG Smear Broad, 0.1 M sodium/potassium tartrate, 10% ethylene glycol, 0.1 M cacodylate pH 5.5. Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Anomalous data with high redundancy were collected at Diamond Light Source beamline I04 using a wavelength of 1.77Å.
Structure determination of WNK3 kinase (monophosphorylated, A-loop exchange)
Monophosphorylated WNK3 kinase domain (a.a. 132-414) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM TCEP, and concentrated to 10 mg/mL. 1 mM adenylyl-imidodiphosphate (AMP-PNP) and 2 mM MgCl2 were added, but not observed in the resulting structure. Crystals were grown at 4°C in 150 nL sitting drops at a 2:1 protein: reservoir solution comprising 20% PEG4000, 10% 2-propanol, 0.1M HEPES pH 7.5. Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04.
Structure determination of WNK3 kinase (AMP-PNP bound, diphosphorylated)
Diphosphorylated WNK3 kinase domain (a.a 133-414) was buffered in 50 mM HEPES pH 7.5, 50 mM NaCl, 0.5 mM TCEP, 10 mM DTT and concentrated to 9.5 mg/mL. A final concentration of 4 mM adenylyl-imidodiphosphate (AMP-PNP) and 10 mM MgCl2 were added. Crystals were grown at 20°C in 150 nL sitting drops at a 1:2 protein: reservoir solution comprising 25% PEG3350, 0.1M bis-tris pH 5.5. Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04.
Structure determination of WNK3 kinase (PP121 bound, diphosphorylated)
Diphosphorylated WNK3 kinase domain (a.a 133-414) was buffered in 50 mM HEPES, 50 mM NaCl, 1 mM TCEP and concentrated to 9 mg/mL. A final concentration of 1 mM PP121 was added. Crystals were grown at 20°C in 150 nL sitting drops at a 1:1 protein: reservoir solution comprising 25% PEG3350, 0.2M magnesium chloride, 0.1M bis-tris pH 5.5. Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04.
Structure determination of WNK1 CCT2 domain
WNK1 CCT2 domain (a.a 1115-1201) was buffered in 20 mM HEPES, 100 mM NaCl and concentrated to 12 mg/mL. Crystals were grown at 20°C in 150 nL sitting drops at a 1:1 protein: reservoir solution comprising 25% PEG3350, 0.2 M ammonium sulfate, 0.1 M bis-tris pH 6.5. Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04-1.
Structure determination of WNK2 CCT1 domain (apo structure)
WNK2 CCT1 domain (a.a 454–549) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP and concentrated to 12 mg/mL. Crystals were grown at 20°C in 150 nL sitting drops at a 2:1 protein: reservoir solution comprising 30% PEG1000, 0.1 M SPG pH 8.0 (succinic acid, phosphate, glycine). Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04-1.
Structure determination of WNK2 CCT1 domain in complex with RFXV-containing peptide
WNK2 CCT1 domain (a.a 454–549) was buffered in 50 mM HEPES pH 7.5, 300 mM NaCl, 1 mM TCEP and concentrated to 12 mg/mL. Crystals were grown at 20°C in 150 nL sitting drops at a 1:1 protein: reservoir solution comprising 20% PEG3350, 0.2 M sodium fluoride, 10% ethylene glycol. Before mounting, crystals were cryo-protected with mother liquor supplemented with an additional 25 % ethylene glycol and vitrified in liquid nitrogen. Diffraction data were collected at Diamond Light Source beamline I04-1.
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