Crystallographic Automation

描述(申请人提供)：在过去的10年里，成像大分子的原子结构对于确定疾病的分子机制和设计治疗方法都是重要的。这导致了人们对确定蛋白质和核苷酸的晶体结构的兴趣增加。对于X射线结晶学研究，初始结晶条件的确定是必不可少的，但对于可溶性蛋白质来说，可能需要数百次试验，对于膜蛋白质来说，可能需要数万次试验。这使得对没有该领域先前经验的研究人员来说，进行这些实验令人望而生畏，并可能阻碍使用结晶学作为主要技术的实验室与专注于功能的实验室之间的合作。该提案要求提供资金，通过从Zinsserna公司(加利福尼亚州北岭)购买一个用于立方脂相(CLP)膜蛋白结晶的机器人和从Formulatrix公司(马萨诸塞州沃尔瑟姆)购买一台自动成像仪来记录结晶反应的时间过程，从而加强范德比尔特大学的结晶自动化。此次收购的两个目标是：1)增强膜蛋白的结晶能力，2)提高自动化结晶设施的能力，以便接触到非结构生物学专业的研究人员。这项提议目前得到范德比尔特大学和范德比尔特大学医学中心的14个实验室的认可，其中4个主要用户和10个次要用户。虽然这些团队已经成功地使用了由蚊子纳米升滴定器组成的晶体自动化，但还没有机器人可以在1000英里内移液输送脂肪相。当地结晶机器人设施的扩展，包括一个旋转立方脂阶段的模块，将对范德比尔特大学由NIH赞助的基础和翻译研究产生广泛的积极影响。此外，将样品运送到异地使用成像设施将销毁这些样品。因此，在校园内监测越来越多的结晶反应的能力将扩大实验室的数量，这些实验室可以在处理人类健康问题时将结构技术作为其技能的一部分。 与健康相关：对大分子结构的测定可以提供对功能和错误功能的基本机制的洞察。为了更好地解决结晶中的难题，我们建议购买一台专为处理膜蛋白而设计的结晶机器人，以及一台自动成像仪。

DESCRIPTION (provided by applicant): 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. HEALTH RELEVANCE: 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.