Molecular placement of experimental electron density: a case study on UDP-galactopyranose mutase

The structure of UDP-galactopyranose mutase, the enzyme responsible for the conversion of UDP-galactopyranose to UDP-galactofuranose, has been solved. The structure solution required the use of two crystal forms and a selenom ethionine variant. Crystal form P2(1) was used to collect a complete MAD da ta set, a native data set and a single-wavelength non-isomorphous selenomet hionine data set. A starting set of MAD phases was then improved by non-cry stallographic averaging and cross-crystal averaging of all P2(1) data. The initial maps were of such low quality that transformation matrices between cells could not be determined. It was therefore assumed that although there were large changes in unit-cell parameters, the molecule occupied the same position in each cell. This starting assumption was allowed to refine duri ng the averaging procedure and did so satisfactorily. Despite a visible inc rease in the quality of the map allowing some secondary-structural elements to be located, the overall structure could not be traced and refined. The rediscovery of the second crystal form, P2(1)2(1)2(1), allowed the collecti on of a native data set to 2.4 Angstrom. Molecular placement of electron de nsity was used to determine the relationship between the two unit cells. In this study, only the already averaged P2(1) experimental density could be placed in the P2(1)2(1)2(1) map. Less extensively density-modified maps did not give a clear solution. The study suggests even poor non-isomorphous da ta can be used to significantly improve map quality. The relationship betwe en P2(1) and P2(1)2(1)2(1) could then be used in a final round of cross-cry stal averaging to generate phases. The resulting map was easily traced and the structure has been refined. The structure sheds important light on a no vel mechanism and is also a therapeutic target in the treatment of tubercul osis.