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.
Additive screen kit
Manufactured by Hampton Research
The Additive screen kit is a laboratory equipment designed for screening and evaluating the performance of various additives in chemical or biological applications. The kit provides the necessary components and tools to conduct controlled experiments and assess the effects of different additives on targeted processes or systems.
Automatically generated - may contain errors
Lab products found in correlation
High throughput crystallization screening kits, by Hampton Research (2 mentions)
Gryphon crystallization robot, by Art Robbins Instruments (2 mentions)
Mosquito crystallization robot, by SPT Labtech (1 mentions)
Index screen, by Hampton Research (1 mentions)
Crystal screen 1 2, by Hampton Research (1 mentions)
5 protocols using additive screen kit
1
Optimized Crystallization of Protein Complexes
2
Structural Characterization of AtTai4 and AtTai4-AtTae4 Complex
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Initial crystallization trials were performed at 293 K by the sitting-drop vapor-diffusion method in a 96-well crystallization plate using various commercially available screening kits. Crystallization drops were prepared by mixing 200 nl purified protein solution and 200 nl reservoir solution using a Mosquito crystallization robot (TTP Labtech). The initial crystals of AtTai4 were optimized at 293 K by varying the concentrations of PEG and salt in the reservoir solution using an Additive Screen kit (Hampton Research). Plate-shaped crystals of AtTai4 were obtained in 33% PEG 6000, 1.5 M lithium chloride, 100 mM sodium acetate. The AtTae4–AtTai4 complex formed thick plate-shaped crystals using MemGold reservoir condition E11 consisting of 35% PEG 400, 0.05 M Tris pH 8.5, 0.05 M sodium sulfate, 0.05 M lithium sulfate.
3
Optimization of TNYR SaPLD Mutant Crystallization
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Screening of crystallisation conditions for the TNYR SaPLD mutant was carried out starting with the known crystallisation condition of wild-type (WT) SaPLD, which was 15% polyethylene glycol MWt-6000 (PEG6K) and 100 mM 2-(N-morpholinoethanesulfonic acid (MES) buffer pH 6.0. The screening was conducted by the hanging drop vapour diffusion method using VDX 24-well plates. Lead conditions from the initial screens were further optimised using the Additive Screen Kit (Hampton Research). Optimised crystallisation drops contained 1 µl protein, 1 µl crystallisation solution (14 to 22% (w/v) PEG6K and 100 mM MES pH 4.0–4.8), and 0.2 µl 5% (w/v) lauryldimethylamine N-oxide (LDAO). Drops were equilibrated at 20°C against 1 ml crystallisation solution in the reservoir. Needle cluster-like crystals appeared within ∼7 days.
4
Optimized Crystallization of Protein Complexes
5
Crystallization of RTA-Peptide Complexes
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Synthesized peptides C9-P2 and C11-P2 (GL Biochem, Shanghai, China) were added into RTA to final concentration 5 mM and incubated for 6 h at 4 °C before crystallization.
Commercially available Crystal Screen 1–2 and Index screen (Hampton Research) were used for crystallization trials in 96-well plates (XtalFinder, XtalQuest Inc., Beijing, China) at 16 °C. The crystals were obtained using the hanging drop vapor-diffusion method, by equilibrating 1 μL of 15 mg/mL RTA-C9-P2 mixture with an equal volume of the reservoir solution (2.8 M sodium acetate, tetrahydrate pH 7.0) (USB, Cleveland, OH, USA). Further optimization was carried out using Additive Screen kit (Hampton Research). Crystals which produced good diffraction quality were grown in 2.8 M sodium acetate tetrahydrate, pH 7.0, 30%–35% glucose. All the crystals were transferred to cryoprotectant (reservoir solution supplemented with 30% glycerol) and flash-cooled with liquid nitrogen. The data were collected at 100 K in a liquid nitrogen stream using beamline 13B1 with a Q315r CCD (ADSC, MAR Research, Norderstedt, Germany) at the Biological Crystallization Facility at National Synchrotron Radiation Research Center (NSRRC), Hsinchu, Taiwan. Data were scaled and merged with ScalePack installed with HKL2000 [31 ].
Commercially available Crystal Screen 1–2 and Index screen (Hampton Research) were used for crystallization trials in 96-well plates (XtalFinder, XtalQuest Inc., Beijing, China) at 16 °C. The crystals were obtained using the hanging drop vapor-diffusion method, by equilibrating 1 μL of 15 mg/mL RTA-C9-P2 mixture with an equal volume of the reservoir solution (2.8 M sodium acetate, tetrahydrate pH 7.0) (USB, Cleveland, OH, USA). Further optimization was carried out using Additive Screen kit (Hampton Research). Crystals which produced good diffraction quality were grown in 2.8 M sodium acetate tetrahydrate, pH 7.0, 30%–35% glucose. All the crystals were transferred to cryoprotectant (reservoir solution supplemented with 30% glycerol) and flash-cooled with liquid nitrogen. The data were collected at 100 K in a liquid nitrogen stream using beamline 13B1 with a Q315r CCD (ADSC, MAR Research, Norderstedt, Germany) at the Biological Crystallization Facility at National Synchrotron Radiation Research Center (NSRRC), Hsinchu, Taiwan. Data were scaled and merged with ScalePack installed with HKL2000 [31 ].
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