Femtosecond nano-crystallography of membrane proteins

描述(申请人提供)：该项目旨在进一步发展用于膜蛋白结构测定的飞秒纳米晶学方法。30%的人类蛋白质组由膜蛋白组成，膜蛋白控制和调节细胞之间的相互作用，调节细胞内和细胞外的运输，也是生物能量转换的主要参与者。它们对人类健康的重要性是压倒性的，目前所有药物中有60%是针对膜蛋白的。飞秒结晶学是一种分析生物大分子X射线结构的新方法，它利用自由电子激光的飞秒X射线脉冲，从蛋白质的完全水合的纳米/微晶中收集数十万个X射线衍射快照。X射线自由电子激光的峰值通量是第三代同步加速器的109倍。虽然X射线脉冲如此强大，以至于它们可以摧毁任何固体材料，但X射线衍射发生在生物分子被摧毁之前。在纳米晶体生长和表征方面的新发展，以及新注射器技术和数据评估方法的开发，使基于该项目的结果的SFX新方法取得了非常快的进展，该项目已发表了16篇论文，其中9篇发表在《科学》杂志上，1篇专利正在申请中。该提案侧重于四个主要目标，其中包括开发新的纳米晶体生长和表征方法(目标1)，创新新的晶体分选和进样技术(目标2)，进一步开发数据分析方法，包括SFX数据的从头算阶段(目标3)，以及利用SFX确定膜蛋白质结构和时间分辨SFX的新途径(目标4)。目标是开启结构生物学的新纪元，SFX被开发并用于解决具有挑战性的膜蛋白质结构。时间分辨SFX的发展将向最终目标提供新的里程碑，以确定膜蛋白在作用中的分子电影。

DESCRIPTION (provided by applicant): This project aims to further develop the method of femtosecond nanocrystallography for the structure determination of membrane proteins. 30% of the human proteome consist of membrane proteins, which control and mediate the interaction between cells, regulate transport in and out of the cells and are also the major players in bioenergy conversion. Their importance for human health is overwhelming, with 60% of all current drugs being targeted to membrane proteins. Femtosecond crystallography is a new method for X-ray structure analysis of biological macromolecules, where hundreds of thousands of X-ray diffraction snapshots are collected from a stream of fully hydrated nano/microcrystals of proteins, using femtosecond X-ray pulses from a Free Electron Laser. The peak flux of an X-ray FEL is 109 times higher than the flux from a 3rd generation Synchrotron. While the X-ray pulses are so strong that they destroy any solid material, X-ray diffraction occurs before the biomolecules are destroyed. New developments in nanocrystal growth and characterization, together with development of new injector technology and data evaluation methods have progressed the new method of SFX at a very fast pace based on results from this project which has led to 16 publications, 9 of them in Nature of Science journals and one patent (pending). The proposal is focused on four major aims which include the development of new methods for nanocrystal growth and characterization (aim 1), innovations on new crystal sorting and Injector technology (aim 2), further development of data analysis methods including de novo phasing of SFX data (aim 3) and determination of membrane protein structures with SFX and new avenues for time-resolved SFX (aim 4). The goal is to open a new era in Structural Biology, where SFX is developed and used to solve challenging membrane protein structures. The development of time resolved SFX will provide new milestones towards the final goal to determine molecular movies of membrane proteins in action.