The TB structural genomics consortium crystallization facility:: towards automation from protein to electron density
The TB structural genomics consortium crystallization facility:: towards automation from protein to electron density
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DOI：
10.1107/s0907444902014282
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发表时间：
2002-10-01
期刊：
影响因子：
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通讯作者：
Earnest, T
中科院分区：
文献类型：
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作者：
Rupp, B;Segelke, BW;Earnest, T
TB（结核病）结构基因组学联盟的结晶设施是NIH赞助的九个P50结构基因组中心之一，为TB联盟成员提供自动结晶，数据收集和基本分子置换（MR）结构解决方案，以最大限度地减少电子密度图的偏差。每天多达10种蛋白质的结晶设置遵循ESTOOL组合筛选方案，使用具有开放式架构的模块化和经济实惠的机器人设计。组件包括屏幕准备、板设置、自动图像采集和分析以及优化设计。一个新的96孔结晶板已被设计为最佳的机器人处理，同时保持方便的手动晶体收获。机器人晶体安装，筛选和数据收集在内部和伯克利的高级光源（ALS）进行。基于MR和基于同源性的结构预测的简单自动化方案自动解决了适度困难的问题。在并行MR中评估多个搜索模型，并且使用CNS对最佳多段刚体细化MR解进行模拟退火扭转角分子动力学，从而在随后的高效偏差去除和图重建协议Shake& wARP的收敛半径内带来甚至边缘MR解，用于生成用于初始重建的电子密度。真实的空间相关图可以快速评估局部结构质量。机器人的模块化设计和使用公共程序的结构解决方案的自动化脚本允许高效的高通量晶体学-以合理的成本。
The crystallization facility of the TB (Tuberculosis) structural genomics consortium, one of nine NIH sponsored P50 structural genomic centres, provides TB consortium members with automated crystallization, data collection and basic molecular replacement (MR) structure solution up to bias minimized electron density maps. Crystallization setup of up to ten proteins per day follows the CRYSTOOL combinatorial screen protocol using a modular and affordable robotic design with an open architecture. Components include screen preparation, plate setup, automated image acquisition and analysis, and optimisation design. A new 96 well crystallization plate has been designed for optimal robotic handling while maintaining ease of manual crystal harvesting. Robotic crystal mounting, screening, and data collection are conducted in-house and at the Advanced Light Source (ALS) in Berkeley. A simple automated protocol based on MR and homology based structure prediction automatically solves modestly difficult problems. Multiple search models are evaluated in parallel MR and the best multi-segment rigid body refined MR solution is subjected to simulated annealing torsion angle molecular dynamics using CNS, bringing even marginal MR solutions within the convergence radius of the subsequent highly effective bias removal and map reconstruction protocol, Shake& wARP, used to generate electron density for initial rebuilding. Real space correlation plots allow rapid assessment of local structure quality. Modular design of robotics and automated scripts using publicly available programs for structure solution allow for efficient high throughput crystallography - at a reasonable cost.