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 by single-particle reconstruction. Cryo-electron tomography of protein nanocrystals has the potential to overcome these obstacles of mainstream structure determination methods. Here we present a data processing scheme that combines routines from X-ray crystallography and new algorithms we 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. We also assess the tolerance of this workflow to the effects of radiation damage. Our 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 datasets, these results recommend distributing the dose over a wide angular range rather than using a finer sampling interval to solve the protein structure.

中文翻译：

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通过模拟评估蛋白质纳米晶体的辐射损坏的断层图解决结构中的挑战

需要结构确定方法来解析构成蛋白质功能基础的原子细节。X射线晶体学已经提供了我们对蛋白质结构的大多数知识，但是受到对大型，有序晶体的需求以及相信息丢失的限制。飞秒串行晶体学，微电子衍射和单颗粒重建的快速发展方法通过允许从纳米晶体或纯化的蛋白质中收集数据来规避了这些限制中的第一个。但是，前两种方法也存在相问题，而许多蛋白质都低于单颗粒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技术有可能克服主流结构确定方法的这些障碍。在这里，我们提出了一种数据处理方案，该方案结合了X射线晶体学的例程和我们开发的新算法，可以解决纳米晶体断层图中的结构。该管道可处理特定于周期性样本的层析成像采样的图像处理挑战，并已使用模拟晶体进行了验证。我们还评估了该工作流程对辐射损伤的耐受性。我们的仿真表明，在更宽的倾斜范围和更小的倾斜增量之间进行权衡，可以方便地合并来自多个断层图的数据，而较小的倾斜增量可以提高相位精度。由于可以通过其他数据集克服相位误差而不是合并误差，因此这些结果建议在较宽的角度范围内分配剂量，而不是使用更小的采样间隔来解决蛋白质结构。