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Gryphon crystallization robot

Manufactured by Art Robbins Instruments
Sourced in United States

The Gryphon crystallization robot is a laboratory instrument designed for automated protein crystallization screening. It systematically creates and monitors crystallization experiments, providing a consistent and efficient method for exploring protein crystallization conditions.

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22 protocols using Gryphon crystallization robot

Initial crystallization screens for all 4 protein complexes were carried out at 20°C with a Gryphon crystallization robot (Art Robbins Instruments) using high-throughput crystallization screening kits (Hampton Research and Qiagen). Extensive manual optimizations were then performed at 20°C using the hanging-drop vapor-diffusion method when proteins were mixed with reservoir solution at 1:1 ratio.
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Initial crystallization screens were carried out using a Gryphon crystallization robot (Art Robbins Instruments) with high-throughput crystallization screening kits (Hampton Research and Qiagen). Extensive manual optimization was then performed at 20°C using the hanging-drop vapor-diffusion method when proteins were mixed with reservoir solutions in 1:1 ratio. The HCA–bSV2C complex was initially crystallized in a condition containing 100 mM sodium cacodylate, pH 6.5, 13% polyethylene glycol (PEG) 3,350, and 200 mM NaCl. The best crystals were obtained in the presence of 0.7% (v/v) 1-butanol, which was identified using an additive screen kit (Hampton Research). The crystals were cryo-protected in the original mother liquor supplemented with 20% (v/v) glycerol and flash-frozen in liquid nitrogen. The HCA–gSV2C complex was originally crystallized as thin plates in a condition composed of 100 mM sodium acetate, pH 4.6, 20% PEG 3,350, and 200 mM ammonium phosphate monobasic. These crystals diffracted poorly. After extensive additive screening and optimization, the best crystals were obtained in the presence of 4% (w/v) polypropylene glycol P 400. The crystals were cryo-protected in the reservoir solution supplemented with 20% (v/v) ethylene glycol and flash-frozen in liquid nitrogen.
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Initial crystallization screens were performed using a Gryphon crystallization robot (Art Robbins Instruments) and high-throughput crystallization screen kits (Hampton Research and QIAGEN). Extensive manual optimizations were performed at 18°C when proteins were mixed with reservoir solution at 1:1 ratio.
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Initial crystallization screens were performed using a Gryphon crystallization robot (Art Robbins Instruments) and high-throughput crystallization screen kits (Hampton Research and QIAGEN). Extensive manual optimizations were performed at 18°C when proteins were mixed with reservoir solution at 1:1 ratio.
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Crystallization trials for the Ric-8A:ΔN31Gαi1:Nb8109:Nb8117:Nb8119 complex were conducted by vapor diffusion using commercially available crystallization screening kits. Sitting drops were set on 96-2 well INTELLI-PLATEs (Art Robbins Instruments) using a Gryphon crystallization robot (Art Robbins Instruments) at 3–10 mg/mL protein complex at a 1:1 v/v ratio with precipitation solution. Initial crystallization conditions were identified from hits on the ShotGun screen (MD1-88 Molecular Dimensions). Further optimization was carried out by grid screening variations in sodium malonate (Hampton Research) concentration and pH and by crystal seeding by hanging drop on 24-well VDXm plates (Hampton Research). Crystal seed stocks were prepared with the Seed Bead kit (Hampton Research) in 1.4 M sodium malonate pH 6.9. 0.9 μl of protein stock was added to 0.6 μl of reservoir and 0.3 μl of crystal seed stock and incubated at 12 °C for a minimum of 1–2 weeks. Optimal crystals were obtained from hanging drops containing 3.6 mg/ml Ric-8A:ΔN31Gαi1:3Nb, 1.4 M sodium malonate pH 6.9 at 12 °C.
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Purified QQQ protein was concentrated to at least 30 mg/mL. Concentrated protein was mixed with 1.5 parts (w/w) of monolein containing 10% (w/w) cholesterol using the syringe reconstitution method (Caffrey and Cherezov, 2009 (link)), to generate a lipidic cubic phase mixture. 25 nL droplets of the mixture were dispensed on glass plates and overlaid with 600 nL of precipitant using a Gryphon crystallization robot (Art Robbins Instruments, Sunnyvale, CA). Crystallization trials were performed in 96-well glass sandwich plates incubated at 16°C. The best crystals were obtained using a precipitant solution consisting of 100 mM Tris (pH 8.5), 100 mM sodium malonate, 30% PEG 400% and 2.5% MPD Crystals were harvested after 3–4 weeks of incubation and flash-frozen in liquid nitrogen without further additives. Figures were prepared using PyMOL and Adobe Illustrator.
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Initial crystallization screening of AfOYE1 (20 mg/mL) was carried out at 18 °C with high-throughput sparse matrix crystallization trails using a Gryphon crystallization robot (Art Robbins Instruments). MRC 96-well two-drop standard plates were adopted with the sitting-drop vapor diffusion method by mixing 0.5 μL protein and 0.5 μL reservoir solution in the initial screening. A 24-well plate was used with the hanging-drop vapor diffusion method by mixing 1 μL protein and 1 μL reservoir solution in manual optimization steps. Well-diffracted crystals were achieved in the condition containing 0.1 M Bis–Tris propane, pH 6.5, 15% (w/v) PEG 3350, and 0.2 M sodium nitrate, after seeding with smaller crystals initially obtained in the same condition. Crystals were cryoprotected in mother liquor supplemented with 25% (v/v) ethylene glycerol and snap-frozen in liquid nitrogen for data collection.
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Protein samples of the D1R-Gs-Compound 1 complex, at the concentration of 20 mg/ml, were reconstituted into lipidic cubic phase (LCP) by mixing with molten lipid (10% (w/w) cholesterol (Sigma), 90% (w/w) 1-(7Z-tetradecenoyl)-rac-Glycerol (MAG7.7, Avanti Polar Lipids) in a mechanical syringe mixer at a ratio of 1:1 (w/w) protein solution to lipid. LCP crystallization trials were performed using Gryphon crystallization robot (Art Robbins Instruments). The 30 nl bolus were covered with 800 nl of precipitant solution. The 96-well glass sandwich plates were incubated at 20 °C. Initial crystal hits were found from MemStart + MemSys HT-96 screen (Molecular Dimensions) with condition 0.1 M NaCl, 0.1 M MgCl2, 0.1 M sodium citrate pH 5.5 and 30% (w/v) PEG400. After optimization, crystals grew in 0.1 M MES pH 6.5, 0.1 M ammonium dibasic citrate, 18–25% (w/w) PEG400, 1 mM TCEP, 10 µM Compound 1 and with or without positive modulator in the precipitant solution. Crystals grew 5–7 days to full size (about 20–30 μm across all three dimensions) and were collected using MiTeGen micromounts and flash-frozen in liquid nitrogen for data collection.
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All protein complexes were isolated by SEC, concentrated to 10 mg/ml and then screened using a Gryphon crystallization robot (Art Robbins Instruments). All hits from sitting drop screens were reproduced and optimized in hanging drops at 25 °C prior to harvesting and freezing. The final crystallization conditions for each complex are as follows: Omalizumab scFV: IgE-Fc3–4 0.02 M Magnesium chloride, 0.1 M HEPES pH 7.5, 22% (w/v) Polyacrylic acid 5100 sodium salt. 813:IgE-Fc3–4: 0.1 M CAPS pH 10.5 40% (v/v) MPD. C02:IgE-Fc3–4: 0.2 M Lithium sulfate, 0.1 M Phosphate citrate pH 4.2 20% (w/v) PEG 1000. HAE:IgE-Fc3–4: 0.1 M Sodium acetate pH 4.6, 8% (w/v) PEG 4000. C02/HAE:IgE-Fc3–4: 0.2 M Lithium sulfate, 0.1 M Sodium acetate pH 4.5, 50% (w/v) PEG 400.
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Initial crystallization screening of the TcdA GTD-AH3 and the TcdA1073-1464-AA6 complexes were carried out at 18°C using a Gryphon crystallization robot (Art Robbins Instruments) with high-throughput sparse matrix screening kits (Hampton Research and Qiagen) using the sitting-drop vapor diffusion method (0.3 µl protein + 0.3 µl reservoir equilibrated against 55 µl reservoir). Crystal optimizations were carried out using the hanging-drop vapor diffusion method at 18°C by mixing equal volume of protein and reservoir solutions. The best crystals of the TcdA GTD-AH3 complex were obtained in a condition containing 0.2 M potassium phosphate and 20% (w/v) PEG 3350. The best crystals of the TcdA1073-1464-AA6 complex were obtained in a condition with 0.2 M ammonium acetate, 0.1 M Bis-Tris, pH 5.7, and 15% PEG 3350. Crystals were cryoprotected in the mother liquor supplemented with 15% (v/v) glycerol and snap frozen in liquid nitrogen for data collection.
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