Femtosecond nano-crystallography of membrane proteins

项目描述（申请人提供）：本项目旨在进一步发展飞秒纳米晶体学方法，用于膜蛋白的结构测定。人体蛋白质组的30%由膜蛋白组成，膜蛋白控制和介导细胞间的相互作用，调节细胞内外的运输，也是生物能量转化的主要参与者。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。飞秒晶体学是一种用于生物大分子x射线结构分析的新方法，其中使用来自自由电子激光器的飞秒x射线脉冲，从完全水合的蛋白质纳米/微晶体流中收集了数十万个x射线衍射快照。x射线自由电子激光器的峰值通量是第三代同步加速器的109倍。虽然x射线脉冲非常强大，可以破坏任何固体物质，但x射线衍射发生在生物分子被破坏之前。纳米晶体生长和表征的新发展，以及新的注入技术和数据评估方法的发展，使SFX的新方法以非常快的速度发展，该项目的结果已经发表了16篇论文，其中9篇发表在Nature of Science期刊上，还有一项专利（正在申请中）。该提案集中在四个主要目标上，包括开发纳米晶体生长和表征的新方法（目标1），创新新的晶体分选和注入技术（目标2），进一步开发数据分析方法，包括SFX数据的重新相位（目标3），以及用SFX测定膜蛋白结构和时间分辨SFX的新途径（目标4）。目标是打开结构生物学的新时代，其中SFX被开发并用于解决具有挑战性的膜蛋白结构。时间分辨SFX的发展将为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.