Substantial advances have been made in the computational design of protein interfaces over the last 20 years. However, the interfaces targeted by design have typically been stable and high‐affinity. Here, we report the development of a generic computational design method to stabilize the weak interactions at crystallographic interfaces. Initially, we analyzed structures reported in the Protein Data Bank to determine whether crystals with more stable interfaces result in higher resolution structures. We found that for 22 variants of a single protein crystallized by a single individual, the Rosetta‐calculated `crystal score' correlates with the reported diffraction resolution. We next developed and tested a computational design protocol, seeking to identify point mutations that would improve resolution in a highly stable variant of staphylococcal nuclease (SNase). Using a protocol based on fixed protein backbones, only one of the 11 initial designs crystallized, indicating modeling inaccuracies and forcing us to re‐evaluate our strategy. To compensate for slight changes in the local backbone and side‐chain environment, we subsequently designed on an ensemble of minimally perturbed protein backbones. Using this strategy, four of the seven designed proteins crystallized. By collecting diffraction data from multiple crystals per design and solving crystal structures, we found that the designed crystals improved the resolution modestly and in unpredictable ways, including altering the crystal space group. Post hoc, in silico analysis of the three observed space groups for SNase showed that the native space group was the lowest scoring for four of six variants (including the wild type), but that resolution did not correlate with crystal score, as it did in the preliminary results. Collectively, our results show that calculated crystal scores can correlate with reported resolution, but that the correlation is absent when the problem is inverted. This outcome suggests that more comprehensive modeling of the crystallographic state is necessary to design high‐resolution protein crystals from poorly diffracting crystals.

中文翻译：

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致力于提高分辨率的蛋白质晶体的计算设计。


过去 20 年来，蛋白质界面的计算设计取得了实质性进展。然而，设计的目标接口通常是稳定且高亲和力的。在这里，我们报告了一种通用计算设计方法的开发，以稳定晶体界面的弱相互作用。最初，我们分析了蛋白质数据库中报告的结构，以确定具有更稳定界面的晶体是否会产生更高分辨率的结构。我们发现，对于由单个个体结晶的单一蛋白质的 22 个变体， Rosetta计算的“晶体分数”与报告的衍射分辨率相关。接下来，我们开发并测试了一种计算设计方案，旨在识别能够提高高度稳定的葡萄球菌核酸酶 (SNase) 变体分辨率的点突变。使用基于固定蛋白质骨架的协议，11 个初始设计中只有一个具体化，这表明建模不准确，迫使我们重新评估我们的策略。为了补偿局部主链和侧链环境的轻微变化，我们随后设计了一组扰动最小的蛋白质主链。使用这种策略，七种设计的蛋白质中有四种结晶。通过收集每个设计的多个晶体的衍射数据并求解晶体结构，我们发现所设计的晶体以不可预测的方式适度地提高了分辨率，包括改变晶体空间群。 事后对 SNase 的三个观察到的空间群进行的计算机分析表明，对于六个变体（包括野生型）中的四个来说，原生空间群的​​得分最低，但该分辨率与晶体得分并不相关，就像在初步结果。总的来说，我们的结果表明，计算出的晶体分数可以与报告的分辨率相关，但当问题反转时，这种相关性就不存在。这一结果表明，需要对晶体状态进行更全面的建模，以从衍射不良的晶体中设计出高分辨率的蛋白质晶体。