DATA COLLECTION FOR MBP_POLYMERASE FUSION PROTEIN

University Of Chicago, Chicago IL

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Abstract

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. Our protein of interest is a human DNA damage repair polymerase, for which a structure has not yet been reported. The polymerase used in the current study was constructed as a fusion with the maltose binding protein (MBP), which increases expression levels and protein solubility. In addition, the presence of the MBP aids molecular replacement. Using this technique, we have successfully purified large quantities of soluble fusion protein, and we have obtained diffraction quality crystals of this polymerase in ternary complex with a DNA substrate and an incoming nucleotide. This polymerase-substrate complex is present in a hexagonal space group with unit cell edges of a = b = 78[unreadable], c = 544[unreadable]. On our home-source 007HF X-ray generator, we have observed diffraction at 2.6[unreadable], however, collection of a complete data set, where we can resolve reflections along the long cell edge, has proven difficult. We have collected several data sets on our home-source x-ray generator, using VariMax HF mirrors and a Saturn92 CCD detector (Figure 1), and on an RAXISIV detector equipped with VariMax HR mirrors for a more collimated beam (Figure 2). Although the CCD detected diffraction at higher resolution, data collection was hindered by beam divergence, coupled with goniometer limits of the CCD. Using the VariMax HR mirrors with the RAXISIV detector decreased beam divergence, but the increased distance decreased spot intensity. Attempting to decrease problems with beam collimation and loss of intensity, we used the mail-in service for the SER-CAT beamlines. Data were collected by Dr. Z. Jin on several crystals. However, he was unable to satisfactorily resolve the reflections along the long cell edge, despite observing diffraction spots to as far as 2.3[unreadable] (Figure 3). We feel that access to the 14-BM-C beamline with its specialized ADSC Quantum 4 CCD detector would allow us to resolve our overlap problem so that we can collect a complete data set at a reasonable resolution.