Global S&T Development Trend Analysis Platform of Resources and Environment
DOI | 10.1126/science.aba0954 |
Advances and challenges in time-resolved macromolecular crystallography | |
Gisela Brändén; Richard Neutze | |
2021-08-27 | |
发表期刊 | Science
![]() |
出版年 | 2021 |
英文摘要 | Macromolecular x-ray crystallography typically provides static snapshots of systems at equilibrium. Advances in time-resolved crystallography have made it possible to capture dynamics in biomolecules: large and small, fast and slow. Brändén and Neutze review techniques and concepts that have emerged from recent work at x-ray free electron laser sources and are now being applied in other settings and to a growing number of biological systems. Despite challenges in analyzing and relating these data to a biological context, experiments in this field have opened new frontiers in temporal and spatial resolution and yielded many new insights into nonequilibrium chemistry and conformational changes in biology. Science , aba0954, this issue p. [eaba0954][1] ### BACKGROUND Conformational changes are essential for the correct functioning of biological macromolecules. Time-resolved x-ray crystallography extends an extremely successful method for the structural determination of biomolecules by incorporating time as a fourth dimension. Time-resolved x-ray diffraction studies are performed at room temperature so as to allow the biological reaction to evolve within crystals. This reaction must also be initiated throughout crystals—and x-ray diffraction data must be collected—at least as rapidly as the fastest time point of interest. Mature structural analysis tools of macromolecular crystallography can then be adapted to allow x-ray diffraction data to be analyzed in terms of time-dependent conformational changes. ### ADVANCES Decades of work using polychromatic x-ray pulses (Laue diffraction) at synchrotron radiation sources laid the experimental foundations underpinning the field of time-resolved macromolecular crystallography. Light-driven biological reactions can be rapidly initiated throughout crystals using short laser pulses and have therefore been a major focus for the field. Serial crystallography was first demonstrated 10 years ago at an x-ray free-electron laser (XFEL). In this approach, x-ray diffraction data are collected from a sequence of microcrystals, typically 10 μm or less in their largest dimension, that are being continuously replaced. X-ray diffraction data from thousands of microcrystals are then merged into a complete dataset. Sample delivery for serial crystallography experiments at an XFEL initially relied on liquid microjets, but many other sample-delivery technologies have since been developed, each with its own strengths and weaknesses. Serial crystallography has overcome many of the technical limitations that constrained time-resolved Laue diffraction and has thereby transformed the field, creating a renaissance of interest in time-resolved macromolecular crystallography. In this Review, we describe how time-resolved x-ray diffraction studies using XFEL pulses a few femtoseconds (10−15 s) in duration have allowed atomic motions in biological samples to be visualized on the time scales at which chemical bonds break or isomerize or at which electrons move. We illustrate the power of time-resolved serial crystallography to yield structural and functional insights on slower time scales by showcasing structural results from two energy-transducing membrane proteins, bacteriorhodopsin and photosystem II, neither of which were amenable to synchrotron-based time-resolved Laue diffraction. We also discuss structural results obtained when using mixing jets to diffuse reactants into microcrystals or when releasing photocaged compounds using a laser flash, which have allowed biological reactions that are not naturally light sensitive to be followed in time. ### OUTLOOK Time-resolved crystallography is transitioning from a highly technical domain of specialists into a flexible approach for elucidating structural and functional insights from macromolecules in their crystalline state. Although serial crystallography was first developed for XFEL-based studies, the recent transfer of time-resolved serial crystallography to synchrotron radiation facilities is critical for the growth of the user community. Nonspecialist user communities will also drive standardized experimental setups that further lower entry barriers for new users. Structural conclusions drawn from XFEL-based studies of ultrafast structural changes should be consolidated by repeating these experiments using lower power density photoexcitation conditions, and data analysis steps—from processing experimental data through to structural interpretation—need to be streamlined. As further structural insights emerge from an increasingly diverse set of macromolecules, it becomes possible to imagine a time when structural results from time-resolved diffraction experiments become as central to understanding a biological reaction as the resting-state structure of a macromolecule is today. ![Figure][2] Schematic illustration of a time-resolved serial crystallography study of a light-sensitive protein. A continuous stream of microcrystals (purple) is injected across a focused x-ray beam (orange) at a synchrotron or XFEL facility. Diffraction data collected from microcrystals photoactivated by a laser pulse (green) are compared against reference diffraction data without photoactivation. Electron density changes (inset: positive difference electron density, blue; negative, yellow) are modeled as changes in protein structure for each time delay between laser and x-ray pulse, providing structural insight into the biological reaction. Conformational changes within biological macromolecules control a vast array of chemical reactions in living cells. Time-resolved crystallography can reveal time-dependent structural changes that occur within protein crystals, yielding chemical insights in unparalleled detail. Serial crystallography approaches developed at x-ray free-electron lasers are now routinely used for time-resolved diffraction studies of macromolecules. These techniques are increasingly being applied at synchrotron radiation sources and to a growing diversity of macromolecules. Here, we review recent progress in the field, including visualizing ultrafast structural changes that guide the initial trajectories of light-driven reactions as well as capturing biologically important conformational changes on slower time scales, for which bacteriorhodopsin and photosystem II are presented as illustrative case studies. [1]: /lookup/doi/10.1126/science.aba0954 [2]: pending:yes |
领域 | 气候变化 ; 资源环境 |
URL | 查看原文 |
引用统计 | |
文献类型 | 期刊论文 |
条目标识符 | http://119.78.100.173/C666/handle/2XK7JSWQ/336648 |
专题 | 气候变化 资源环境科学 |
推荐引用方式 GB/T 7714 | Gisela Brändén,Richard Neutze. Advances and challenges in time-resolved macromolecular crystallography[J]. Science,2021. |
APA | Gisela Brändén,&Richard Neutze.(2021).Advances and challenges in time-resolved macromolecular crystallography.Science. |
MLA | Gisela Brändén,et al."Advances and challenges in time-resolved macromolecular crystallography".Science (2021). |
条目包含的文件 | 条目无相关文件。 |
除非特别说明,本系统中所有内容都受版权保护,并保留所有权利。
修改评论