Clabbers Max T B, Gonen Tamir
Struct Dyn. 2025 May 13;12(3):034702. doi: 10.1063/4.0000755. eCollection 2025 May.
Inelastic scattering poses a significant challenge in electron crystallography by elevating background noise and broadening Bragg peaks, thereby reducing the overall signal-to-noise ratio. This is particularly detrimental to data quality in structural biology, as the diffraction signal is relatively weak. These effects are aggravated even further by the decay of the diffracted intensities as a result of accumulated radiation damage, and rapidly fading high-resolution information can disappear beneath the noise. Loss of high-resolution reflections can partly be mitigated using energy filtering, which removes inelastically scattered electrons and improves data quality and resolution. Here, we systematically compared unfiltered and energy-filtered microcrystal electron diffraction data from proteinase K crystals, first collecting an unfiltered dataset followed directly by a second sweep using the same settings but with the energy filter inserted. Our results show that energy filtering consistently reduces noise, sharpens Bragg peaks, and extends high-resolution information, even though the absorbed dose was doubled for the second pass. Importantly, our results demonstrate that high-resolution information can be recovered by inserting the energy filter slit. Energy-filtered datasets showed improved intensity statistics and better internal consistency, highlighting the effectiveness of energy filtering for improving data quality. These findings underscore its potential to overcome limitations in macromolecular electron crystallography, enabling higher-resolution structures with greater reliability.
非弹性散射在电子晶体学中构成了重大挑战，它会提高背景噪声并展宽布拉格峰，从而降低整体信噪比。这对结构生物学中的数据质量尤其不利，因为衍射信号相对较弱。由于累积的辐射损伤导致衍射强度衰减，这些影响会进一步加剧，快速衰减的高分辨率信息可能会消失在噪声之下。使用能量过滤可以部分缓解高分辨率反射的损失，能量过滤可以去除非弹性散射电子，提高数据质量和分辨率。在这里，我们系统地比较了蛋白酶K晶体的未过滤和能量过滤的微晶电子衍射数据，首先收集一个未过滤的数据集，然后直接使用相同设置进行第二次扫描，但插入了能量过滤器。我们的结果表明，即使第二次扫描时吸收剂量增加了一倍，能量过滤仍能持续降低噪声、锐化布拉格峰并扩展高分辨率信息。重要的是，我们的结果表明，通过插入能量过滤狭缝可以恢复高分辨率信息。能量过滤数据集显示出更好的强度统计和更高的内部一致性，突出了能量过滤对提高数据质量的有效性。这些发现强调了其克服大分子电子晶体学局限性的潜力，能够获得更高分辨率且可靠性更高的结构。
