Since ATPase crystals can be flash-cooled rather easily, we attempted to study intermediates formed during ATP hydrolysis by quenching the reaction at several time increments after introducing ATP to the crystal. Once the crystal is flash-cooled, monochromatic methods of data collection which require several hours can be used to acquire better quality and higher resolution data compared to Laue diffraction data. Crystals of one particular mutant of ATPase was used to perform these experiments for which the rate of hydrolysis is significantly reduced to 100-1000 sec in solution. We developed methods to quench the reaction, store and transfer crystals based on the advice from Hakon Hope (UC Davis). We observed that the crystals began to crack after reacting for ~15 min at room temperature, which was also confirmed in the diffraction data. We repeated the experiment at lower temperature (~4l C) and collected data sets just before cracking was observed. The data were of excellent quality and virtually 100% complete, however, the difference maps showed no indication of structural changes. We believe the onset of cracking may be associated with the hydrolysis step in the reaction and that a large structural change may, in fact, occur.

Agency
National Institute of Health (NIH)
Institute
National Center for Research Resources (NCRR)
Type
Biotechnology Resource Grants (P41)
Project #
5P41RR001209-17
Application #
5222646
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
17
Fiscal Year
1996
Total Cost
Indirect Cost
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