PROJECT SUMMARY This supplement requests a mosquito Xtal3 crystallization robot for the parent grant (GM144578), which will reduce the required sample amounts for structure determination projects to 10% of what is currently needed. This will allow the PI to complete a larger scope of work, as protein purification is the most time-consuming step in X-ray crystallography. The robot will also increase reproducibility of crystallization trials and allow to expand the scope of the parent grant to include structure determination of larger complexes with low expression yields. The parent grant investigates how the cell nucleus is bi-directionally transported and positioned in a cell cycle specific manner, a process that is important for brain and muscle development. This is underscored by the fact that human disease mutations of proteins engaged in the transport of the nucleus cause severe brain and muscle development diseases, including spinal muscular atrophy, which is the most common genetic cause of death in infants. Yet, it is unknown how teams of opposite polarity motors collaborate to achieve correct timing, directionality and velocity of transport. We plan: 1) To establish how motility of the motor complex dynein is modulated by dynein adapter/cargo complexes and by the opposite polarity motor complex kinesin-1. 2) to establish a structural basis for recognition of the cell nucleus as cargo by dynein adapters. 3) To establish whether the opposite polarity motors dynein and kinesin-1 are recruited in a cooperative manner to the nucleus. Currently, the PI’s structural studies are focused on minimal protein complexes with truncated fragments that are expressed in E. coli with high yields. The PI has successfully reconstituted larger complexes with full-length dynein adapters and intact motors. While the structure determination of larger complexes was already suggested as an alternative approach in the parent grant, it is currently not feasible due to the lower yields of the insect cell expression system. The robot will allow to carry out crystallization trials with smaller drops compared to manual pipetting, which will reduce the sample requirements to 10%. This will increase the impact of all three aims of the parent grant since the PI will be able to expand her structural studies to intact complexes, which will reveal a greater level of mechanistic insights compared to truncated complexes. This is particularly important for the proposed aims which plan to establish the activation and cargo-recognition mechanism of BicD2, as well as how bi-directional transport of the opposite polarity motors kinesin-1 and dynein is regulated by adapter proteins. Our study serves as a model system to understand how cargo adapters regulate the motility and directionality of cargo transported bi-directionally by both dynein and kinesin-1, which is important as these motors facilitate a vast number of cellular transport events that are essential for chromosome segreg...