The N-terminal domain of SARS-CoV-2 nsp110-126, containing residues 10 to 126 and named nsp1 throughout the manuscript, was expressed, purified and crystallised as previously described [6 (link)]. The crystallisation condition used is Index 71 (Cat. No.: HR2-944-71) 0.1 M BIS-TRIS pH 6.5, 0.2 M NaCl and 25% w/v Polyethylene glycol 3350 from Hampton Research, Aliso Viejo, CA, USA.
Fragment hits 5E11, 10B6, and 11C6 were obtained from the Maybridge Ro3 library, while 7H2 was purchased from Molport, Beacon, NY, USA (Cat. No.: HY-30379). Each of them was dissolved in DMSO-d6 (Sigma, St. Louis, MO, USA; CAS Number: 2206-27-1) at a concentration of 200 mM as a stock solution. An amount of 2 μL of each stock solution was mixed with 8 μL of the final buffer (10 mM HEPES pH 7.6 and 300 mM NaCl) (Sigma, St. Louis, MO, USA; CAS Number: 7365-45-9 and 7647-14-5), giving a final concentration of 40 mM fragment containing 20% DMSO-d6. Each fragment solution (1.5 μL) was added into approximately 1 μL of crystallisation drops, making a final fragment concentration of 24 mM and approximately 12% DMSO-d6. These drops were incubated at room temperature for 4–5 h followed by crystal harvesting using loops, cryo-cooled in liquid nitrogen and stored in pucks for sample storage and shipment.
Fragment hits 5E11, 10B6, and 11C6 were obtained from the Maybridge Ro3 library, while 7H2 was purchased from Molport, Beacon, NY, USA (Cat. No.: HY-30379). Each of them was dissolved in DMSO-d6 (Sigma, St. Louis, MO, USA; CAS Number: 2206-27-1) at a concentration of 200 mM as a stock solution. An amount of 2 μL of each stock solution was mixed with 8 μL of the final buffer (10 mM HEPES pH 7.6 and 300 mM NaCl) (Sigma, St. Louis, MO, USA; CAS Number: 7365-45-9 and 7647-14-5), giving a final concentration of 40 mM fragment containing 20% DMSO-d6. Each fragment solution (1.5 μL) was added into approximately 1 μL of crystallisation drops, making a final fragment concentration of 24 mM and approximately 12% DMSO-d6. These drops were incubated at room temperature for 4–5 h followed by crystal harvesting using loops, cryo-cooled in liquid nitrogen and stored in pucks for sample storage and shipment.
Free full text: Click here
