Abstract
WbdD is a bifunctional kinase/methyltransferase that is responsible for regulation of lipopolysaccharide O antigen polysaccharide chain length in Escherichia coli serotype O9a. Solving the crystal structure of this protein proved to be a challenge because the available crystals belonging to space group I23 only diffracted to low resolution (>95% of the crystals diffracted to resolution lower than 4 Å and most only to 8 Å) and were non-isomorphous, with changes in unit-cell dimensions of greater than 10%. Data from a serendipitously found single native crystal that diffracted to 3.0 Å resolution were non-isomorphous with a lower (3.5 Å) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 Å resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 Å resolution data set to build a crude model of WbdD is desribed. Using this crude model as a mask to cut out the 3.5 Å resolution electron density yielded a successful molecular-replacement solution of the 3.0 Å resolution data set. The resulting map was used to build a complete model of WbdD. The hydration status of individual crystals appears to underpin the variable diffraction quality of WbdD crystals. After the initial structure had been solved, methods to control the hydration status of WbdD were developed and it was thus possible to routinely obtain high-resolution diffraction (to better than 2.5 Å resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations.
Highlights
The last two decades have seen a steady improvement of the infrastructure and techniques that are used to express, purify and crystallize proteins
We designed, cloned and expressed a new construct, WbdD556, that was purified in the same way as WbdD600
After the structure of WbdD556 had been solved as described above, the model was used to solve the structure of the 4.2 Alow-resolution data set (Table 2) in order to inves- single kinase domain failed
Summary
The last two decades have seen a steady improvement of the infrastructure and techniques that are used to express, purify and crystallize proteins. The actual structure-solution process has become increasingly streamlined (Winn et al, 2011; Adams et al, 2002; Winter, 2010). Structures can literally be solved by a single keystroke and the process is routine in many cases (Oke et al, 2010). The remaining hurdle is obtaining reproducible high-quality crystals. This represents a particular problem for challenging targets such as protein complexes, membrane proteins and post-translationally modified eukaryotic proteins. Even proteins that are anticipated to be routine prove to be difficult and their study can identify approaches for a priori challenging cases
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