Crystallization screenings were performed using high-throughput techniques in a NanoDrop robot and Innovadyne SD-2 microplates (Innovadyne Technologies, Inc.) with screening PACT Suite and JCSG Suite (Qiagen), JBScreen Classic 1 to 4 (Jena Bioscience), Morpheus and MIDASplus (Molecular Dimensions) and Crystal Screen I&II, SaltRX and Index HT (Hampton Research). The conditions that produced crystals were optimized with a sitting-drop vapor-diffusion method at 291 K by mixing 1μl of protein solution and 1 μl of precipitant solution, equilibrated against 150 μl of precipitant solution in the reservoir chamber. For p6CΔ31, the best crystals were obtained in a crystallization condition containing 0.1 M Bis–Tris Propane pH 7.0 and 1.3 M di-Ammonium Tartrate (Supplementary Figure S2B ). For p6CΔ20, the best crystals were obtained in a crystallization condition containing 10% w/v PEG 8000; 20% v/v ethylene glycol; 0.02 M 1,6-hexanediol; 0.02 M 1-butanol; 0.02 M (RS)-1,2-propanediol; 0.02 M 2-propanol; 0.02 M 1,4-butanediol; 0.02 M 1,3-propanediol and 0.1 M MES/imidazole pH 6.5. In both cases, protein concentration was assayed at the concentration of 9–10 mg/ml.
Jbscreen classic 1 to 4
Manufactured by Jena Biosciences
JBScreen Classic 1 to 4 are screening kits designed to identify optimal crystallization conditions for proteins and other biomolecules. The kits contain a variety of different buffers, salts, and precipitants that can be used to systematically screen for suitable crystallization conditions.
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2 protocols using jbscreen classic 1 to 4
1
High-throughput Protein Crystallization Screening
2
Crystallization and X-ray Structural Analysis of PBP1
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Crystallization screenings were performed using high-throughput crystallization techniques in a NanoDrop robot using Innovadyne SD-2-microplates (Innovadyne Technologies, Inc.) and screening using JBScreen PACT++, JBScreen Classic 1 to 4, JBScreen JCSG++ 1 to 4 (Jena Bioscience), Crystal Screen, Crystal Screen 2, SaltRx HT, Index HT (Hampton Research), and Wizard Cryo (Rigaku) protocols. The conditions that produced crystals were optimized by sitting-drop vapor diffusion method at 290 K by mixing 1 µL of protein solution and 1 µL of precipitant solution, equilibrated against 150 µL of precipitant solution in the reservoir chamber. Crystals of PBP1 were obtained in 2.1 M dl -malic acid pH 7.0 at a concentration of 10 mg mL−1. Crystals of PBP1·penicillin G complex were obtained by co-crystallization in 1.6 M sodium citrate at a concentration of 11 mg mL−1. The complex with piperacillin was obtained by soaking the PBP1 crystals in the crystallization solution containing 2 mM of piperacillin for 30 min. The complex with pentaglycine was obtained by soaking the PBP1 crystals overnight in the crystal solution containing 0.45 mM of pentaglycine.
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