Over the past 10 years, it has become clear that imaging the atomic structures of macromolecules is important for both determining the molecular mechanisms of disease and to design therapeutics. This has lead to an increase in interest in determining crystal structures of proteins and nucleotides. For X-ray crystallographic studies, the identification of initial crystallization conditions is essential, but can take hundreds of trials for a soluble protein, and tens of thousands of trials for a membrane protein. This makes approaching these experiments daunting for researchers without prior experience in the field, and can inhibit collaborations between laboratories that use crystallography as a primary technique and laboratories focused on function. The proposal requests funds to enhance the crystallographic automation at Vanderbilt University through the purchase one robot for the crystallization of membrane proteins in cubic lipid phases (CLP) from Zinsserna (Northridge, CA) and one automated imager from Formulatrix (Waltham, MA) to document the time- course of crystallization reactions. The two goals of this purchase are: 1) to enhance the capabilities for crystallization of membrane proteins, and 2) to improve the capacity of the automated crystallization facility to allow outreach to researchers that do not specialize in structural biology. This proposal is currently endorsed by 14 laboratories at Vanderbilt University and the Vanderbilt University Medical Center, with 4 major users and 10 minor users. While these groups have had success using crystallographic automation consisting of a Mosquito nano-liter drop setter, there is no robot that can pipette the lipid phase within 1,000 miles. The expansion of the local crystallization robotic facility to include a module that pippettes the cubic lipid phase will have a broad positive impact on NIH-sponsored basic and translational research at Vanderbilt. Moreover, transportation of samples off-site to using imaging facilities will destroy these samples. Thus the on-campus ability to monitor an increased number of crystallization reactions will expand the number of laboratories that can use structural techniques as a part of their repertoire as they approach problems of human health.
The determination of structures of macromolecules can provide insight into the basic mechanisms of function and mis-function. To better approach difficult problems in crystallization, we propose to purchase a crystallization robot designed to work with membrane proteins, and an automated imager.
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