Author: Linlin Zhang; Daizong Lin; Yuri Kusov; Yong Nian; Qingjun Ma; Jiang Wang; Albrecht von Brunn; Pieter Leyssen; Kristina Lanko; Johan Neyts; Adriaan de Wilde; Eric J. Snijder; Hong Liu; Rolf Hilgenfeld
Title: Alpha-ketoamides as broad-spectrum inhibitors of coronavirus and enterovirus replication Document date: 2020_2_10
ID: 7n8p9okf_56
Snippet: Freshly prepared CVB3 3C pro at a concentration of 21.8 mg·mL -1 was incubated with 5 mM 11a pre-dissolved in 100% DMSO at room tempature for 1 h. Some white precipitate appeared in the mixture. Afterwards, the sample was centrifuged at 13,000 x g for 20 min at 4 ℃. The supernatant was subjected to crystallization trials using the following, commercially available kits: Sigma TM (Sigma-Aldrich), Index TM , and PEG Rx TM (Hampton Research). Sin.....
Document: Freshly prepared CVB3 3C pro at a concentration of 21.8 mg·mL -1 was incubated with 5 mM 11a pre-dissolved in 100% DMSO at room tempature for 1 h. Some white precipitate appeared in the mixture. Afterwards, the sample was centrifuged at 13,000 x g for 20 min at 4 ℃. The supernatant was subjected to crystallization trials using the following, commercially available kits: Sigma TM (Sigma-Aldrich), Index TM , and PEG Rx TM (Hampton Research). Single rod-like crystals were detected both from the Index TM screen, under the condition of 0.1 M MgCl2 hexahydrate, 0.1 M Bis-Tris, 25% PEG 3,350, pH 5.5, and from the Sigma TM screen at 0.2 M Li2SO4, 0.1 M Tris-HCl, 30% PEG 4,000, pH 8.5. Crystal optimization was performed by using the vapor-diffusion sitting-drop method, with 1 μL CVB3 3C pro -inhibitor complex mixed with 1 μL precipitant solution, and equilibration against 500 μL reservoir containing 0.1 M Tris-HCl, 0.2 M MgCl2, pH 8.5, and PEG 3,350 varied from 22% to 27%. Another optimization screen was also performed against a different reservoir, 0.1 M Tris-HCl, 0.2 M MgCl2, pH range from 7.5 to 8.5, and PEG 4,000 varied from 24% to 34%. Crystals were fished from different drops and protected by cryo-protectant solution consisting of the mother liquor and 10% glycerol. Subsequently, the crystals were flash-cooled with liquid nitrogen.
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