Nextal tubes cryos suite

The TrSOX gene (GeneBank: ACM06094.1) was cloned in a pMA5-Pxyl-trsox plasmid and kept in our laboratory. The HisTrap HP Ni-column was purchased from GE Healthcare; AmiconUltra centrifugal filter tube (10 kDa) was from Merck Millipore; Thin-layer chromatography (TLC) plates (Merck, 105626, 105729, 105721 and 105554) were purchased from Merck Millipore; Q5 Site-Directed Mutagenesis Kit was from NEB Inc.; NeXtal Tubes JCSG Core Suites I-IV, NeXtal Tubes Classics Suite, NeXtal Tubes Classics II Suite, and NeXtal Tubes Cryos Suite were from Qiagen; and Pierce BCA Protein Assay Kit was from Thermo Scientific. N-methyl or alkaloid substrates were from MACKLIN and ALADDIN. Other reagents and chemicals were purchased from local companies and were of superior analytical grade.

In our previous work, the expression of TrSOX was promoted with a single mutant-S320R (23 (link)). The crystallization of TrSOX (S320R) was screened using the hanging-drop vapor diffusion method with NeXtal Tubes JCSG Core Suites I-IV, NeXtal Tubes Classics Suite, NeXtal Tubes Classics II Suite, and NeXtal Tubes Cryos Suite (Qiagen). Diffraction datasets were collected at the Beamline 17U, Shanghai Synchrotron Radiation Facility (SSRF). The crystal structure of TrSOX (S320R) was resolved by molecular replacement and further refinement using the CCP4 Program Suite 6.1.3. The models of native TrSOX and the mutants were built and refined using the “Mutate Protein” module in the software Discovery Studio 2019, and the “Receptor-Ligand Interactions” module was employed to screen the substrate pocket and to investigate the interaction between FAD and the residues in the coenzyme pocket, and the “CDOCKER” module of Discovery Studio 2019 was employed to simulate the binding of substrates in the substrate pocket.