4ax8
Medium resolution structure of the bifunctional kinase- methyltransferase WbdDMedium resolution structure of the bifunctional kinase- methyltransferase WbdD
Structural highlights
FunctionWBDD_ECOLX Regulates the length of the LPS O-antigen polysaccharide chain. Stops the polymerization of the chain by phosphorylating and then methylating the phosphate on the terminal sugar. This terminal modification is essential for export of the O-antigen across the inner membrane. WbdD is also required for correct localization of the WbdA mannosyltransferase.[1] [2] [3] Publication Abstract from PubMedWbdD 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 A and most only to 8 A) 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 A resolution were non-isomorphous with a lower (3.5 A) resolution selenomethionine data set. Here, a strategy for improving poor (3.5 A resolution) initial phases by density modification and cross-crystal averaging with an additional 4.2 A 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 A resolution electron density yielded a successful molecular-replacement solution of the 3.0 A 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 A resolution). This novel and facile crystal-dehydration protocol may be useful for similar challenging situations. Crystallization, dehydration and experimental phasing of WbdD, a bifunctional kinase and methyltransferase from Escherichia coli O9a.,Hagelueken G, Huang H, Harlos K, Clarke BR, Whitfield C, Naismith JH Acta Crystallogr D Biol Crystallogr. 2012 Oct;68(Pt 10):1371-9. Epub 2012 Sep 18. PMID:22993091[4] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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