Protein crystallization, data collection and structure
determination
The home-grown crystals were initially screened manually using a
laboratory-made kit at room temperature using a 96-well plate with
diffused vapor diffusion. A 0.8 μL aliquot of XynA protein solution (8.0
mg/mL, 20 mM Tris-HCl pH 8.0, 287 mM NaCl) was mixed with a 0.8 μL
reservoir solution and equilibrated with a 100 μL reservoir solution.
Twenty-four hours later, the initial XynA crystals were obtained in 28%
PEG600 (w/v), 0.1 M CaCl2 and 0.1 M MES pH 6.0. The
optimized crystallization conditions were 24% PEG600 (w/v), 0.2 M
CaCl2, 0.1 M MES pH 6.0, crystal length reaches 2 mm.
29% PEG600 (w/v), 0.1 M CaCl2, 0.1 M MES pH 6.0 and
25% glycerol were used as refrigerating protectors. Individual crystals
were installed on a nylon loop and immediately cooled with liquid
nitrogen prior to data collection. The individual crystals are installed
on a nylon loop and immediately cooled with liquid nitrogen. In Shanghai
Synchrotron Radiation Facility (SSRF-BL19U1) diffraction data was
collected through X-ray diffraction and finally processed using HKL2000
(Otwinowski & Minor, 1997). We use PDB 1V0L as a model to obtain the
initial model. Based on the initial model, we use COOT to accurately
modify the structure, and then use CCP4 for refinement. Finally, repeat
the above steps until the structural requirements are met (Emsley,
Lohkamp, Scott, & Cowtan, 2010; McCoy et al., 2007). XynA finally
obtained a 2.3Å resolution crystal structure. The crystallization
conditions of the XynA E182A/E280A were the same as those of XynA, and
the structure of the XynA E182A/E280A was obtained with a resolution of
2.9 Å. Information about the protein crystal structure is provided inTable 1 . Conservation analysis of protein structure was carried
out through the ConSurf Server website and PyMOL software was
used to prepare images.