Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation.
Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation.
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DOI：
10.1107/s2059798321002369
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发表时间：
2021-05-01
期刊：
影响因子：
--
通讯作者：
Jensen GJ
中科院分区：
文献类型：
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作者：
Peck A;Yao Q;Brewster AS;Zwart PH;Heumann JM;Sauter NK;Jensen GJ
提出了一种从蛋白质纳米晶体断层图中求解结构的数据处理管道，并使用模拟晶体进行了验证。评估了该工作流对辐射损伤的鲁棒性，以研究最佳数据收集策略。需要结构测定方法来解析蛋白质功能背后的原子细节。x射线晶体学为我们提供了蛋白质结构的大部分知识，但由于需要大而有序的晶体和相位信息的丢失，x射线晶体学受到了限制。快速发展的连续飞秒晶体学、微电子衍射和单粒子重建方法通过从纳米晶体或纯化蛋白质中收集数据，绕过了第一个限制。然而，前两种方法也存在相问题，而许多蛋白质低于单粒子重建所需的分子量阈值。蛋白质纳米晶体的低温电子断层扫描有可能克服这些主流结构测定方法的障碍。本文提出了一种数据处理方案，该方案结合了x射线晶体学的常规方法和已经开发的用于从纳米晶体层析成像中求解结构的新算法。该管道处理周期性标本层析成像采样特有的图像处理挑战，并使用模拟晶体进行验证。该工作流程对辐射损伤影响的容忍度也进行了评估。模拟结果表明，在更宽的倾斜范围和更小的倾斜增量之间进行权衡，以方便合并来自多个层析图的数据，并提高相位精度。由于相位误差，而不是合并误差，可以用额外的数据集来克服，这些结果建议在一个宽角度范围内分配剂量，而不是使用更细的采样间隔来解决蛋白质结构。
A data-processing pipeline to solve structures from tomograms of protein nanocrystals is presented and validated using simulated crystals. The robustness of this workflow to radiation damage is assessed to investigate optimal data-collection strategies. Structure-determination methods are needed to resolve the atomic details that underlie protein function. X-ray crystallography has provided most of our knowledge of protein structure, but is constrained by the need for large, well ordered crystals and the loss of phase information. The rapidly developing methods of serial femtosecond crystallography, micro-electron diffraction and single-particle reconstruction circumvent the first of these limitations by enabling data collection from nanocrystals or purified proteins. However, the first two methods also suffer from the phase problem, while many proteins fall below the molecular-weight threshold required for single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure-determination methods. Here, a data-processing scheme is presented that combines routines from X-ray crystallography and new algorithms that have been developed to solve structures from tomograms of nanocrystals. This pipeline handles image-processing challenges specific to tomographic sampling of periodic specimens and is validated using simulated crystals. The tolerance of this workflow to the effects of radiation damage is also assessed. The simulations indicate a trade-off between a wider tilt range to facilitate merging data from multiple tomograms and a smaller tilt increment to improve phase accuracy. Since phase errors, but not merging errors, can be overcome with additional data sets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.