RADIATION DAMAGE AS A FUNCTION OF WAVELENGTH

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This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Radiation damage is a serious concern for Anomalous Dispersion phasing experiments because of the increased absorption by the sample at the typical wavelength of interest for MAD or SAD experiments. It has been found that data collection at the absorption edge inflection point and the remote wavelength in 10 degrees wedges allowed structure solution in the case of vinculin, where conventional three-wavelength MAD experiments and SAD at the maximum f¿¿ energy failed. Experiments were carried out to determine a) if the above results are valid for other proteins and b) what is the critical factor which facilitates structure solution in such cases: simultaneous data collection at all wavelengths in wedges, or avoiding data collection at the maximum f'' wavelength. The experiments were carried out on crystals of the hypothetical protein (NP_356412.1) from Agrobacterium tumefaciens and a putative glucosamine-fructose-6-phosphate aminotransferase (16423107) from Salmonella typhimurium: Successive MAD and SAD experiments were collected from several crystals until substantial diffraction damage (substantial decrease of diffraction spots at high resolution was observed. The quality of the phases decreased steadily as the total dose absorbed by the crystals increased. In the case of two-wavelength MAD experiments the electron density maps could still be interpreted after substantial radiation damage as long as the data were collected in wedges. However, when the two wavelengths were collected consecutively, structure solution failed at a lower total absorbed do