STP10 was crystallized in lipidic cubic phase (LCP). To prepare lipidic cubic phase for crystallization trials, the protein was supplemented with 100 mM D-glucose before mixing with a 80% monoolein (Sigma-Aldrich) 20% cholesterol mixture, in 1:1.5 protein to lipid/cholesterol ratio (w/w) using a syringe lipid mixer. For crystallization, 50 nl of the meso phase was mixed with 1000 nl of crystallization buffer for each condition on glass sandwich plates using a Gryphon robot (Art Robbins Instruments). Tiny crystals appeared after one day at 20 °C. These crystals diffracted to ~10 Å at Diamond Light Source beamline I24. The addition of various additives and detergents were used to optimize crystals and the final optimized crystallization screen contained 0.1 M NaCitrate pH 4.5, Ammonium dihydrogen phosphate (75–150 mM), DMSO (5–12%), and PEG400 from 25–35%. This gave crystals with a size of approximately 70 × 10 × 30 μm. The crystals were collected using dual thickness micromounts (MiTeGen) and immediately flash frozen in liquid nitrogen. The final datasets were collected at Diamond Light Source beamline I24 using a wavelength of 0.9686 Å.
Gryphon robot
Manufactured by Art Robbins Instruments
The Gryphon robot is a programmable benchtop robotic system designed for laboratory use. It is capable of performing a range of automated liquid handling tasks with precision and accuracy. The Gryphon robot can be programmed to aspirate, dispense, and transfer liquids between various labware, such as microplates, tubes, and reservoirs.
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6 protocols using gryphon robot
1
STP10 Protein Crystallization in LCP
2
Crystallization of CckARec Protein
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Protein solubilised in SEC buffer (see above) was crystallised using the sitting-drop vapor diffusion method. Sets of 3-drop MRC plates were prepared with a Gryphon robot (Art Robbins Instruments). CckARec was crystallised at three concentrations 20, 10, and 5 mg/mL, with a crystallization mixture consisting of 0.15 M KSCN, 0.1 M Na Cacod 6.5 pH, 20% v/v PEG 600 (Clear Strategy II-D5) at room temperature.
3
Crystallization of SpOatAC Protein
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SpOatAC was concentrated to 42 mg·mL-1 using an Amicon Ultra-15 centrifugal filter (30 kDa MWCO; Millipore (Canada) Ltd., Etobicoke, ON) at 4,000 × g and 4 oC, followed by centrifugation (15,000 × g, 10 min, 4 oC) to remove any insoluble material. The concentrated protein sample was used in the MCSG Crystallization Suite sparse matrix crystallization screens 1 to 4 (Microlytic North America Inc., Burlington, MA). Crystallization screening using the sitting drop vapor diffusion method was setup using a Gryphon robot (Art Robbins Instruments, Sunnyvale, CA) with 1 µL drops of protein and a protein to reservoir ratio of 1:1. Large single diffraction quality crystals appeared after one week of incubation at 21 oC in 0.1 M HEPES:NaOH pH 7.5, 1.2 M sodium citrate tribasic; and 2.4 M sodium malonate pH 7. Crystal screening of selenomethionine (SeMet) labeled OatA was carried out as described above and large single crystals were grown in 2.4 M sodium malonate pH 7. To produce SpOatAC in complex with MeS, crystals grown in 1.8 M NaH2PO4/K2HPO4, pH 8; 0.1 M HEPES:NaOH pH 7.5, 1.4 M sodium citrate tribasic were soaked in mother liquor containing 1.2 M sodium citrate tribasic and 250 mM MSF for 24 hours.
4
Crystallization of SUC1 in Lipidic Cubic Phase
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SUC1 was crystallized in the lipidic cubic phase. Purified protein was mixed with monoolein (Sigma) in a 1:1.5 protein-to-lipid ratio using a syringe mixer. For crystallization, 50 nl of the meso phase were mixed with 1000 nl of crystallization buffer containing 43.5% polyethylene glycol 400 (v/v), 0.25 M ammonium dihydrogen phosphate and 0.15 M bis-tris propane (pH 6.3) on glass sandwich plates using a Gryphon robot (Art Robbins Instruments). The lipidic cubic phase crystals appeared after 1–2 days and grew to full size of 100 × 10 × 30 µm at 19 °C within 1 week. The crystals were collected using Dual Thickness MicroMounts LD (MiTeGen) and flash frozen in liquid nitrogen.
5
Crystallisation of DgcR Variants
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Crystallisation attempts were performed using vapour diffusion method prepared in 3-drop MRC plates by Gryphon robot (Art Robbins Instruments) with DgcR (wild-type or I-site variant AxxA) at a concentration of 10 mg/mL (280 μM) in 20 mM Tris pH 8.0, 20 mM NaCl, 5 mM MgCl2 and 1 mM DTT at 18 ˚C. For DgcR’ crystallisation, 3’dGTP was added at a final concentration of 2 mM. After 3 days, crystals could be observed in 0.2 M Magnesium sulphate, 20% PEG 3350 from condition C8 of PEG/Ion HT crystallisation kit (Hampton Research). DgcR’* was crystallised by the same protocol, but with BeF3- treatment prior to the crystallisation set-up. After 7 days crystals were observed in a condition composed by 0.3 M Magnesium chloride hexahydrate, 0.3 M calcium chloride dehydrate, 1.0 M imidazole, MES monohydrate (acid), pH 6.5, EDO_P8K, 40% v/v ethylene glycol, 20% w/v PEG 8000 present in condition A2 from Morpheus I crystallisation kit (Molecular Dimensions). Crystallisation of DgcR in the inhibited conformation (DgcR_inh) was achieved by the presence of 2.0 mM c-di-GTP. Crystals appeared after 5 days in 0.2 M potassium thiocyanate, 0.1 M Tris pH 7.5, 25% PEG 2000 MME, condition optimised from H11 of Index HT crystallisation kit (Hampton Research). Crystals were frozen in liquid nitrogen and stored in a transport Dewar prior to data collection.
6
Crystallization of Diguanylate Cyclase
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Crystallization attempts were performed using vapour diffusion method prepared in 3-drop MRC plates by Gryphon robot (Art Robbins Instruments) with DgcR (wild-type or I-site mutant AxxA) at a concentration of 10 mg/mL (280 μM) in 20 mM Tris pH 8.0, 20 mM NaCl, 5 mM MgCl2 and 1 mM DTT at 18 ˚C. For DgcR_nat crystallisation, 3'dGTP was added at a final concentration of 2 mM. After 3 days, crystals could be observed in 0.2 M Magnesium sulfate, 20 % PEG 3350 from condition C8 of PEG/Ion HT crystallization kit (Hampton Research). DgcR_act was crystallised by the same protocol, but with BeF3-treatment prior to the crystallisation set-up. After 7 days crystals were observed in a condition composed by 0.3 M Magnesium chloride hexahydrate, 0.3 M calcium chloride dehydrate, 1.0 M imidazole, MES monohydrate (acid), pH 6.5, EDO_P8K, 40% v/v ethylene glycol, 20 % w/v PEG 8000 present in condition A2 from Morpheus I crystallization kit (Molecular Dimensions). Crystallization of DgcR in the inhibited conformation (DgcR_inh) was achieved by the presence of 2.0 mM c-di-GTP. Crystals appeared after 5 days in 0.2 M potassium thiocyanate, 0.1 M Tris pH 7.5, 25% PEG 2000 MME, condition optimized from H11 of Index HT crystallization kit (Hampton Research). Crystals were frozen in liquid nitrogen and stored in a transport Dewar prior to data collection.
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