title: Phasing in the presence of severe site-specific radiation damage through dose-dependent modelling of heavy atoms.
abstract: The case of a brominated RNA crystal structure determination in which standard three-wavelength MAD phasing was unsuccessful because of fast X-ray-induced debromination was reinvestigated [Ennifar et al. (2002), Acta Cryst. D58, 1262-1268]. It was found that if the data are kept unmerged and if a dose-stamp is associated with each reflection measurement, dose-dependent occupancies can be refined for the Br atoms. Such a parametrization has been implemented in the macromolecular phasing program SHARP. Refining such dose-dependent occupancies on an unmerged data set gave a dramatic improvement, even for SAD phases from only the first wavelength (peak), and resulted in a good electron-density map after solvent flattening. The adverse effect of radiation damage has been turned into a beneficial one. The crucial difference is made by the use of unmerged data: phasing power is generated through the intensity differences of symmetry-related reflections recorded at different doses, i.e. corresponding to different states of the X-ray-induced debromination. This approach should prove useful in all situations of experimental phasing where site-specific radiation damage occurs unavoidably and undesirably and not only in cases in which radiation damage is purposely being created in order to demonstrate its potential usefulness.
authors.affiliation: Laboratoire de Cristallographie, Ecole Polytechnique Fédérale de Lausanne, EPFL-FSB-IPMC-LCR, Bâtiment BSP, CH-1015 Lausanne, Switzerland. marc.schiltz@epfl.ch
authors.forename: M
authors.initials: M
authors.lastname: Schiltz
authors.forename: P
authors.initials: P
authors.lastname: Dumas
authors.forename: E
authors.initials: E
authors.lastname: Ennifar
authors.forename: C
authors.initials: C
authors.lastname: Flensburg
authors.forename: W
authors.initials: W
authors.lastname: Paciorek
authors.forename: C
authors.initials: C
authors.lastname: Vonrhein
authors.forename: G
authors.initials: G
authors.lastname: Bricogne
mesh_terms: D001665:Binding Sites; D018360:Crystallography, X-Ray / Q000379:methods*; D004220:Disulfides / Q000737:chemistry; D004307:Dose-Response Relationship, Radiation; D008958:Models, Molecular; D011485:Protein Binding; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D012313:RNA / Q000737:chemistry; D012984:Software; D013223:Statistics as Topic; D014965:X-Rays
chemical_list: D004220:Disulfides; D011506:Proteins; D012313:RNA