Gene to Structure for $10K (GTS10K)

基因到结构仅需 1 万美元 (GTS10K)

• 批准号: 7089035
• 负责人: LANCE J STEWART
• 金额: $ 396.46万
• 依托单位: EMERALD BIOSTRUCTURES
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2010-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7089035
• 关键词: X ray crystallography; biomedical resource; cooperative study; functional /structural genomics; protein structure; proteomics

Description: The field of structural genomics aims to characterize the structure-function relationships of all proteins by the experimental elucidation of the three dimensional atomic structures of representative members of protein families combined with the accurate computation of models for closely sequence related proteins. The path from gene to structure for eukaryotic proteins is riddled with challenges including the error-prone isolation of gene clones from cDNA libraries, poor yields in protein expression and purification, and time lost between the set-up of crystallization experiments and X-ray diffraction analysis of putative crystals. To address these challenges, we propose the development of integrated instruments, methods and software that will substantially increase the success rate while reducing the time, effort, materials, and cost required for protein structure determination. Among these new developments will be: (1) Rosetta-guided design and gene synthesis of expression optimized DNA sequences encoding proteins that are guided towards more facile purification and improved crystallogenesis; (2) nano-volume microfluidic crystallization devices that embody the major protein crystallization methods allowing extensive crystallization screening of precious protein samples and their complexes; and (3) a tunable in-house MAD X-ray source which will allow in situ room-temperature crystal X-ray diffraction screening and final anomalous dispersion diffraction data collection for rapid structure determination. Starting in year 3, these new technologies will be validated through the planned structure determination and public release of over 550 eukaryotic protein targets belonging to families with high relevance to cancer biology and therapy, including kinases, transcription factors, and metabolic enzymes. The dissemination of the proposed instrumentation, methods and software will allow researchers to pursue protein X-ray crystal structures at or below a total cost of $10,000 per eukaryotic protein structure.

描述：结构基因组学领域旨在通过实验阐明蛋白质家族代表性成员的三维原子结构并结合紧密序列相关蛋白质的模型的精确计算来表征所有蛋白质的结构-功能关系。真核蛋白质从基因到结构的道路充满了挑战，包括从 cDNA 文库中容易出错的基因克隆分离、蛋白质表达和纯化的产量低、以及结晶实验设置和假定晶体的 X 射线衍射分析之间的时间损失。为了应对这些挑战，我们建议开发集成的 仪器、方法和软件将大大提高成功率，同时减少蛋白质结构测定所需的时间、精力、材料和成本。这些新进展包括：（1）Rosetta指导的编码蛋白质的表达优化DNA序列的设计和基因合成，这些序列可引导更容易的纯化和改进的晶体发生； (2) 纳米微流控结晶装置，体现了主要的蛋白质结晶方法，可以对珍贵的蛋白质样品及其复合物进行广泛的结晶筛选； (3) 可调谐的内部 MAD X 射线源，可进行原位室温晶体 X 射线衍射筛选和最终的反常色散衍射数据收集，以实现快速结构测定。从第 3 年开始，这些新技术将通过计划的结构测定和公开发布超过 550 个属于与癌症生物学和治疗高度相关的家族的真核蛋白靶标（包括激酶、转录因子和代谢酶）来得到验证。所提出的仪器、方法和软件的传播将使研究人员能够以每个真核蛋白质结构 10,000 美元或低于 10,000 美元的总成本来研究蛋白质 X 射线晶体结构。