Eliminating Critical Systematic Errors In Structural Biology With Next-Generation Simulation

项目摘要/摘要 大分子结晶学中的数据收集会受到严重的系统误差的影响，这些误差会阻止 在许多系统上成功收集数据，最终会限制结果结构的准确性。创造 能够解释这些误差的模拟技术将在三个方面产生重大影响：1)解决 新结构通过更好地解释辐射损伤，这是失败的异常的80%的原因 分相尝试，2)通过模拟非同构来改进多晶平均，这将打开 在信噪比中获得任意增益的途径，3)区分激烈竞争的替代解释，如 通过使用更真实的溶剂模型创建模拟，以确定结合配体的存在或不存在。至 从同步加速器走向“无损伤数据”，我们将从校准辐射损伤曲线开始。 模型和DBP样本。使用这些曲线，我们将结合辐射损害的真实3D模型 将非长方体晶体(RADDOSE 3D)放入我们的衍射图像模拟器(MLFSOM)以产生3D剂量 沿晶体的分布和照明贴图。这将导致新一代波长- 晶体的依赖吸收系数，以补充现有的吸收校正。在光束线处， 我们将使用锥束在线x射线吸收射线照相术和2D图来测量晶体的3D图 光束的轮廓。这些进展将使我们能够在开放的情况下产生零剂量外推值 格式，这解释了实验晶体和光束几何。为了改善多晶体平均，我们将 首先描述非同构如何随湿度、辐射损伤和 功能状态。通过对经典的非同构的Crick和Magdoff模拟进行更新 对于复杂性，我们将开发一种奇异值分解方法来对非同构进行参数化。vbl.使用 从这个分析中得到的修正，我们将修正多晶体中存在的非同构 实验，能够确定新的结构，包括使用序列收集的那些 下一代光源的结晶学。启用增强的模拟以实现可靠的解释 实验数据，我们将利用新的溶剂模型在大分子结晶学和小角X- 光线散射。我们的工作将创建比较溶剂密度与替代溶剂密度的标准方案 并定量评估每种模拟情况与真实情况进行比较的可能性 大分子结晶学或SAXS数据。除了区分不同的对 实验数据，改进溶剂模型将加强对大分子如何影响的理解 并与其表面附近的其他分子相互作用。总体而言，我们期待着消除这些问题的好处 关键的系统误差对方法开发和功能研究都具有变革性。

PROJECT SUMMARY/ABSTRACT Data collection in macromolecular crystallography is subject to significant systematic errors that prevent successful data collection on many systems and, ultimately, limit the accuracy of resulting structures. Creating simulation technologies that can account for these errors will have significant impact on three fronts: 1) solving new structures by better accounting for radiation damage, which is responsible for 80% of failed anomalous phasing attempts, 2) improving multi-crystal averaging by simulating non-isomorphism, which will open the gateway to arbitrary gains in signal-to-noise, 3) discriminating hotly contested alternative interpretations such as the presence or absence of a bound ligand, by creating simulations with more realistic solvent models. To move towards “damage-free data” from a synchrotron, we will start by calibrating radiation damage curves on model and DBP samples. Using these curves we will incorporate realistic 3D models of radiation damage to non-cuboid crystals (RADDOSE 3D) into our diffraction image simulator (MLFSOM) to yield a 3D Dose Distribution and Illumination map along the crystal. This will result in a new generation of wavelength- dependent absorption factors for the crystal to complement existing absorption corrections. At the beamline, we will measure a 3D map of the crystal using cone beam online x-ray absorption radiography and a 2D map of the beam profile. These advances will allow us to generate zero-dose extrapolation values, in an open format, that account for experimental crystal and beam geometry. To improve multi-crystal averaging, we will begin by characterizing how non-isomorphism varies as a function of humidity, radiation damage, and functional state. By updating the classic “Crick and Magdoff” simulations of non-isomorphism with increasing complexity, we will develop a singular value decomposition approach to parameterize non-isomorphism. Using the corrections derived from this analysis, we will correct the non-isomorphism present in multi-crystal experiments, enabling the determination of novel structures, including those collected using serial crystallography at next-generation light sources. To enable enhanced simulation for robust interpretation of experimental data, we will leverage new solvent models in macromolecular crystallography and small angle X- ray scattering. Our work will create standard protocols for comparing solvent density to alternative interpretations and to quantitatively assess how likely each simulated situation is compared to the real macromolecular crystallography or SAXS data. In addition to distinguishing between different interpretations of the experimental data, improving solvent models will enhance understanding of how macromolecules influence and interact with other molecules near their surface. Collectively, we expect the benefits of eliminating these critical systematic errors be transformative to both methods development and functional studies.